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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 Ceará | 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.


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.


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.


Friedrich J.L.R.,Federal University of Rio Grande do Sul | Pena F.P.,Federal University of Rio Grande do Sul | Garcia-Galan C.,ICP CSIC | Fernandez-Lafuente R.,ICP CSIC | And 2 more authors.
Journal of Chemical Technology and Biotechnology | Year: 2013

Background: In this work two immobilized preparations of lipase (EC 3.1.1.3) B from Candida antarctica (CALB) were compared as biocatalysts in the synthesis of ethyl butyrate, a short-chain esters with fruity notes. Commercial Novozym 435 and CALB immobilized on styrene-divinylbenzene beads (MCI-CALB) were tested for esterification reactions. Central composite design and response surface methodology were used to optimize the reaction temperature, substrate molar ratio, enzyme content, and the added water. Results: The two enzymatic preparations presented different optimal conditions concerning ethyl butyrate production, with higher yields of conversion around 85% in 1.5 h being achieved. However, MCI-CALB presented productivities 1.6 times higher than Novozym 435. The main difference between the biocatalysts was in relation to operational stability during batch reuse experiments, in which MCI-CALB retained 80% of its initial activity after eight batches, while Novozym 435 retained only 20% under the same conditions. Conclusion: It was verified that variations in the protocols for enzyme immobilization causes different optimal conditions for the esterification reaction. These are very interesting results because reaction times were short, producing high conversion yields and productivities considering the mass of biocatalyst used. © 2012 Society of Chemical Industry.


Martins A.B.,Federal University of Rio Grande do Sul | Schein M.F.,Federal University of Rio Grande do Sul | Friedrich J.L.R.,Federal University of Rio Grande do Sul | Fernandez-Lafuente R.,ICP CSIC | And 2 more authors.
Ultrasonics Sonochemistry | Year: 2013

The influence of low-frequency ultrasound (40 kHz) in the esterification reaction between acetic acid and butanol for flavor ester synthesis catalyzed by the commercial immobilized lipase B from Candida antarctica (Novozym 435) was evaluated. A central composite design and the response surface methodology were used to analyze the effects of the reaction parameters (temperature, substrate molar ratio, enzyme content and added water) and their response (yields of conversion in 2.5 h of reaction). The reaction was carried out using n-hexane as solvent. The optimal conditions for ultrasound-assisted butyl acetate synthesis were found to be: temperature of 46 °C; substrate molar ratio of 3.6:1 butanol:acetic acid; enzyme content of 7%; added water of 0.25%, conditions that are slightly different from those found using mechanical mixing. Over 94% of conversion was obtained in 2.5 h under these conditions. The optimal acid concentration for the reaction was determined to be 2.0 M, compared to 0.3 M without ultrasound treatment. Enzyme productivity was significantly improved to around 7.5-fold for each batch when comparing ultrasound and standard mechanical agitation. The biocatalyst could be directly reused for 14 reactions cycles keeping around 70% of its original activity, while activity was virtually zeroed in the third cycle using the standard mixing system. Thus, compared to the traditional mechanical agitation, ultrasound technology not only improves the process productivity, but also enhances enzyme recycling and stability in the presence of acetic acid, being a powerful tool to improve biocatalyst performance in this type of reaction. © 2013 Elsevier B.V. All rights reserved.


Garcia-Galan C.,ICP CSIC | Barbosa O.,ICP CSIC | Barbosa O.,Industrial University of Santander | Fernandez-Lafuente R.,ICP CSIC
Enzyme and Microbial Technology | Year: 2013

The enzyme glutamate dehydrogenase (GDH) from Escherichia coli is a hexameric protein. The stability of this enzyme was increased in the presence of Li+ in concentrations ranging from 1 to 10mM, 1M of sodium phosphate, or 1M ammonium sulfate. A very significant dependence of the enzyme stability on protein concentration was found, suggesting that subunit dissociation could be the first step of GDH inactivation. This effect of enzyme concentration on its stability was not significantly decreased by the presence of 10mM Li+. Subunit crosslinking could not be performed using neither dextran nor glutaraldehyde because both reagents readily inactivated GDH. Thus, they were discarded as crosslinking reagents and GDH was incubated in the presence of polyethyleneimine (PEI) with the aim of physically crosslinking the enzyme subunits. This incubation does not have a significant effect on enzyme activity. However, after optimization, the PEI-GDH was found to almost maintain the full initial activity after 2h under conditions where the untreated enzyme retained only 20% of the initial activity, and the effect of the enzyme concentration on enzyme stability almost disappeared. This stabilization was maintained in the pH range 5-9, but it was lost at high ionic strength. This PEI-GDH composite was also much more stable than the unmodified enzyme in stirred systems. The results suggested that a real adsorption of the PEI on the GDH surface was required to obtain this stabilizing effect. A positive effect of Li+ on enzyme stability was maintained after enzyme surface coating with PEI, suggesting that the effects of both stabilizing agents could not be exactly based on the same mechanism. Thus, the coating of GDH surface with PEI seems to be a good alternative to have a stabilized and soluble composite of the enzyme. © 2013 Elsevier Inc.


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.


Barbosa O.,University of Tolima | Ortiz C.,Industrial University of Santander | Berenguer-Murcia A.,University of Alicante | Torres R.,Ecopetrol SA | And 2 more authors.
Biotechnology Advances | Year: 2015

In this review, we detail the efforts performed to couple the purification and the immobilization of industrial enzymes in a single step. The use of antibodies, the development of specific domains with affinity for some specific supports will be revised. Moreover, we will discuss the use of domains that increase the affinity for standard matrices (ionic exchangers, silicates). We will show how the control of the immobilization conditions may convert some unspecific supports in largely specific ones. The development of tailor-made heterofunctional supports as a tool to immobilize-stabilize-purify some proteins will be discussed in deep, using low concentration of adsorbent groups and a dense layer of groups able to give an intense multipoint covalent attachment. The final coupling of mutagenesis and tailor made supports will be the last part of the review. © 2015 Elsevier Inc.


Poppe J.K.,Federal University of Rio Grande do Sul | Garcia-Galan C.,ICP CSIC | Matte C.R.,Federal University of Rio Grande do Sul | Fernandez-Lafuente R.,ICP CSIC | And 2 more authors.
Journal of Molecular Catalysis B: Enzymatic | Year: 2013

In this work two immobilized preparations of lipase (EC 3.1.1.3) B from Candida antarctica (CALB) were compared for the synthesis of fatty acid methyl esters (FAME) using soybean oil. Commercial Novozym 435 (CALB-435) and CALB immobilized on styrene-divinylbenzene beads (CALB-MCI) were tested for the transesterification reactions. Central composite design (CCD) and response surface methodology (RSM) were used to optimize the reaction parameters, substrate molar ratio, enzyme content, and the added amount of water, on the initial reaction rate as response. The biocatalysts showed different optimal conditions for the production of FAME. For CALB-435, optima conditions were 5.6:1 molar ratio methanol:oil, 25% enzyme, and 5.44% of added water, while for MCI-CALB, these optima were 3:1 molar ratio methanol:oil, 25% enzyme, and 1.18% of added water, resulting in initial reaction rates of 51.47 mmol L-1 h-1, and 57 mmol L-1 h-1 of FAME, respectively. Conversions of 93.38% using CALB- 435, and 99.03% using CALB-MCI were obtained after 72 h of reaction under the optimized conditions. Repeated batches of reaction were carried out to test the operational stability of biocatalysts, with both preparations keeping around 70% of their initial activity after eight batches. © 2013 Elsevier B.V.


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.

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