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Manoel E.A.,Federal University of Rio de Janeiro | dos Santos J.C.S.,ICP CSIC | dos Santos J.C.S.,Federal University of Ceara | Freire D.M.G.,Programa de Pos Graduacao em Bioquimica | And 2 more authors.
Enzyme and Microbial Technology | Year: 2015

The lipases from Thermomyces lanuginosus and Pseudomonas cepacia have been immobilized on octyl and cyanogen bromide (CNBr) agarose beads. The immobilization on octyl-agarose is slowed with increasing ionic strength, while the immobilization on CNBr is not significantly affected by the ionic strength. The inhibition of the immobilized preparations with diethyl p-nitrophenylphosphate (D- pNPP) was analyzed. The inhibition was more rapid using octyl-lipase preparations than using covalent preparations, and the covalent preparations were much more sensitive to the reaction medium. The addition of detergent increased the inhibition rate of the covalent preparation while an increase on the ionic strength produced a slowdown of the inhibition rate by D- pNPP for both lipases. The effect of the medium on the activity versus fully soluble substrate (methyl mandelate) was in the same direction. The octyl preparations presented a slight decrease in activity when comparing the results using different concentrations of sodium phosphate buffer (between 0.025 and 1. M), while the CNBr preparations suffered drastic drops in its activity at high ionic strength. The results confirm that the lipases immobilized on octyl agarose presented their open form stabilized while the covalent preparation maintains a closing/opening equilibrium that may be modulated by altering the medium. © 2015 Elsevier Inc. Source


Martins A.B.,Federal University of Rio Grande do Sul | Graebin N.G.,Federal University of Rio Grande do Sul | Lorenzoni A.S.G.,Federal University of Rio Grande do Sul | Fernandez-Lafuente R.,ICP CSIC | And 2 more authors.
Process Biochemistry | Year: 2011

In this paper is described the optimization of the esterification reaction of butyl acetate synthesis catalyzed by Candida antarctica lipase B (Novozym 435). The reaction parameters temperature, substrate molar ratio, enzyme content, and added water, and their responses measured as conversion yields, were evaluated using central composite design and response surface methodology. The best acid concentration for the reaction without enzyme inactivation was determined to be 0.3 M. The optimal conditions for butyl acetate synthesis were found to be temperature of 40 °C; substrate molar ratio of 3:1 butanol:acetic acid; enzyme content of 7.5% of substrate wt.; added water 0.25% of substrate wt. Under these conditions, over 90% of conversion was obtained in 2.5 h. Enzyme reuse was tested performing three different treatments before each batch: washing the enzyme system with either n-hexane or water, or suspending the immobilized enzyme in water for 24 h. Direct enzyme reuse or washing with water produced a rapid decrease on enzyme activity, while washing with n-hexane allowed enzyme to be reused for 6 reactions cycles keeping around 70% of its activity. This fast and high yield of conversion represents a large improvement to previously reported results. © 2011 Elsevier Ltd. All rights reserved. Source


De Abreu L.,Federal University of Rio Grande do Sul | Fernandez-Lafuente R.,ICP CSIC | Rodrigues R.C.,Federal University of Rio Grande do Sul | Volpato G.,Institute of Education | Ayub M.A.Z.,Federal University of Rio Grande do Sul
Journal of Molecular Catalysis B: Enzymatic | Year: 2014

Lipase from Staphylococcus warneri EX17 (SWL) was purified and immobilized via interfacial adsorption using the hydrophobic supports Octyl-sepharose, Immobead 150, and MCI GEL CHP20P. The purity of the obtained immobilized biocatalysts, Octyl-SWL, Immobead-SWL, and MCI-SWL, was evaluated by SDS-PAGE and their thermal and solvent stability were tested. Results indicated that the intensity of the interaction between the lipase and the support surface interferes with the properties of the immobilized enzyme. The immobilized preparations Octyl-SWL and MCI-SWL were stable in the presence of 50% butanol, ethanol, n-hexane, isopropanol, and methanol. Containing only 8 mg g -1 of enzyme in relation to the support, Octyl-SWL and MCI-SWL preparations catalyzed the synthesis of ethyl butyrate in a 24 h reaction, showing conversions of 28% (51.3 mmol mg-1), and 35.6% (65.2 mmol mg-1), respectively. These results indicate that Octyl-SWL and MCI-SWL preparations present very high specific activities. © 2013 Elsevier B.V. All rights reserved. Source


Poppe J.K.,Federal University of Rio Grande do Sul | Fernandez-Lafuente R.,ICP CSIC | Rodrigues R.C.,Federal University of Rio Grande do Sul | Ayub M.A.Z.,Federal University of Rio Grande do Sul
Biotechnology Advances | Year: 2015

Lipases are being extensively researched for the production of biodiesel as a "silver bullet" in order to avoid the drawbacks of the traditional alkaline transesterification. In this review, we analyzed the main factors involved in the enzymatic synthesis of biodiesel, focusing in the choice of the immobilization protocol, and the parameters involved in the choice and configuration of the reactors. An extensive discussion is presented about the advantages and disadvantages of each type of reactor and their mode of operation. The current scenario of the market for enzymatic biodiesel and some future prospects and necessary developments are also briefly presented. © 2015 Elsevier Inc. Source


Paludo N.,Federal University of Rio Grande do Sul | Alves J.S.,Federal University of Rio Grande do Sul | Altmann C.,Federal University of Rio Grande do Sul | Ayub M.A.Z.,Federal University of Rio Grande do Sul | And 2 more authors.
Ultrasonics Sonochemistry | Year: 2015

In this work, the combined use of ultrasound energy and molecular sieves was investigated for the synthesis of ethyl butyrate, ester with mango and banana notes, catalyzed by the immobilized lipase from Thermomyces lanuginosus (Lipozyme TL-IM). Initially, the best concentrations of biocatalysts (35%) and butyric acid (0.7 M) were tested using ultrasound as an alternative to mechanical agitation. The amount of acid in the reaction could be increased by 2-fold when compared to previous works where mechanical agitation was used. In the next step, substrate molar ratio and reaction temperature were optimized and the best conditions were at their lowest levels: 1:1 (acid:alcohol), and 30 °C, reaching 61% of conversion in 6 h. Molecular sieves (3 Å) were added to optimized reaction medium in order to remove the formed water and improve the maximum yield. The reaction yield increased 1.5 times, reaching 90% of conversion in 6 h, when 60 mg of molecular sieves per mmol of butyric acid was used. Finally, the reuse of Lipozyme TL-IM for the ultrasound-assisted synthesis of ethyl butyrate was verified for 10 batches, without any appreciable loss of activity, whereas in systems using mechanical agitation, the biocatalyst was completely inactivated after 5 batches. These results suggest that the combined use of ultrasound and molecular sieves greatly improve esterification reactions by stabilizing the enzyme and increasing yields. © 2014 Elsevier B.V. All rights reserved. Source

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