Navegando por Autor "Peña, Sara Arana"

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Coimmobilization of different lipases: Simple layer by layer enzyme spatial ordering
(Elsevier, 2020-02-15) Peña, Sara Arana; Rios, Nathalia Saraiva; Sanchez, Carmen Mendez; Lokha, Yuliya; Carballares, Diego; Gonçalves, Luciana Rocha Barros; Lafuente, Roberto Fernandez
This paper shows the step by step coimmobilization of up to five different enzymes following two different orders in the coimmobilization to alter the effect of substrate diffusion limitations. The enzymes were the lipases A and B from Candida antarctica, the lipases from Rhizomocur miehei and, Themomyces lanuginosus and the phospholipase Lecitase Ultra. The utilized strategy was a layer by layer immobilization, coating the immobilized enzymes with polyethylenimine followed by the crosslinking of the enzyme and PEI with glutaraldehyde to prevent enzyme release, and them adding a new lipase layer. The use of previously inactivated biocatalysts (using diethyl p-nitrophenylphosphate) permitted to visualize the immobilization of each enzyme layer, which was later confirmed by SDS-PAGE. This also confirmed the successful and complete covalent crosslinking of the glutaraldehyde treated enzyme layers. Activity of the combibiocatalysts was followed using diverse substrates. The protocol was successful and permitted to immobilize in an ordered way the 5 different enzymes in a down-up distribution
Effects of enzyme loading and immobilization conditions on the catalytic features of lipase from pseudomonas fluorescens immobilized on Octyl-Agarose beads
(Frontiers, 2020-02-28) Peña, Sara Arana; Rios, Nathalia Saraiva; Navarro, Diego Carballares; Sanchez, Carmen Mendez; Lokha, Yuliya; Gonçalves, Luciana Rocha Barros; Lafuente, Roberto Fernandez
The lipase from Pseudomonas fluorescens (PFL) has been immobilized on octyl-agarose beads under 16 different conditions (varying pH, ionic strength, buffer, adding some additives) at two different loadings, 1 and 60 mg of enzyme/g of support with the objective of check if this can alter the biocatalyst features. The activity of the biocatalysts versus p-nitrophenyl butyrate and triacetin and their thermal stability were studied. The different immobilization conditions produced biocatalysts with very different features. Considering the extreme cases, using 1 mg/g preparations, PFL stability changed more than fourfolds, while their activities versus pNPB or triacetin varied a 50–60%. Curiously, PFL specific activity versus triacetin was higher using highly enzyme loaded biocatalysts than using lowly loaded biocatalysts (even by a twofold factor). Moreover, stability of the highly loaded preparations was higher than that of the lowly loaded preparations, in many instances even when using 5°C higher temperatures (e.g., immobilized in the presence of calcium, the highly loaded biocatalysts maintained after 24 h at 75°c a 85% of the initial activity, while the lowly loaded preparation maintained only 27% at 70°C). Using the highly loaded preparations, activity of the different biocatalysts versus pNPB varied almost 1.7-folds and versus triacetin 1.9-folds. In this instance, the changes in stability caused by the immobilization conditions were much more significant, some preparations were almost fully inactivated under conditions where the most stable one maintained more than 80% of the initial activity. Results suggested that immobilization conditions greatly affected the properties of the immobilized PFL, partially by individual molecule different conformation (observed using lowly loaded preparations) but much more relevantly using highly loaded preparations, very likely by altering some enzyme-enzyme intermolecular interactions. There is not an optimal biocatalyst considering all parameters. That way, preparation of biocatalysts using this support may be a powerful tool to tune enzyme features, if carefully controlled
Immobilization of lipases via interfacial activation on hydrophobic supports: Production of biocatalysts libraries by altering the immobilization conditions
(Elsevier, 2021-02-15) Peña, Sara Arana; Rios, Nathalia Saraiva; Carballares, Diego; Gonçalves, Luciana Rocha Barros; Lafuente, Roberto Fernandez
Lipases A and B from Candida antarctica (CALA, CALB), and those from Candida rugosa (CRL) and Rhizomucor miehei (RML) have been immobilized on octyl agarose beads under 16 different conditions. The activities of the biocatalysts versus different substrates were analyzed, as well as their thermal stabilities at pH 7.0. All CALB and CRL preparations showed very similar properties, except the CRL biocatalysts prepared at pH 9. Under these conditions the free enzyme was partially inactivated. Immobilized CALA showed some significant differences in activity depending on the immobilization conditions when using esters of mandelic acid as substrates (the activities of the different preparations differed by more than a 2-fold factor), while when using the other substrates the differences were minimal. However, immobilized RML enzyme greatly alters its activity depending on the immobilization conditions, reaching differences up to 4–5 fold in both activity and stability. It is remarkable that the effect of one immobilization variable depends on the substrates, and that there are strong interactions between the different immobilization variables. Thus, this immobilization method was very robust (producing almost identical functional biocatalysts independently from the immobilization conditions) using CRL or CALB, while the immobilization conditions must be carefully controlled using RML to have reproducible results
Influence of phosphate anions on the stability of immobilized enzymes. Effect of enzyme nature, immobilization protocol and inactivation conditions
(Elsevier, 2020-08) Kornecki, Jakub F.; Carballares, Diego; Sterlinga, Roberto Morellon; Siar, El Hocine; Kashefi, Saeid; Chafiaa, Mazri; Peña, Sara Arana; Rios, Nathalia Saraiva; Gonçalves, Luciana Rocha Barros; Lafuente, Roberto Fernandez
A destabilizing effect at pH 7 of sodium phosphate on several lipases immobilized via interfacial activation is shown in this work. This paper investigates if this destabilizing effect is extended to other inactivation conditions, immobilization protocols or even other immobilized enzymes (ficin, trypsin, β-galactosidase, β-glucosidase, laccase, glucose oxidase and catalase). As lipases, those from Candida antarctica (A and B), Candida rugosa and Rhizomucor miehei have been used. Results confirm the very negative effect of 100 mM sodium phosphate at pH 7.0 for the stability of all studied lipases immobilized on octyl agarose, while using glutaraldehyde-support the effect is smaller (still very significant using CALA) and in some cases the effect disappeared (e.g., using CALB). The change of the pH to 5.0 or 9.0, or the addition of 1 M NaCl reduced the negative effect of the phosphate in some instances (e.g., at pH 5.0, this negative effect is only relevant for CALB). Regarding the other enzymes, only the monomeric β-galactosidase from Aspergillus oryzae is strongly destabilized by the phosphate buffer. This way, the immobilization protocol and the inactivation conditions strongly modulate the negative effect of sodium phosphate on the stability of immobilized lipases, and this effect is not extended to other enzymes
Use of polyethylenimine to produce immobilized lipase multilayers biocatalysts with very high volumetric activity using octyl-agarose beads: Avoiding enzyme release during multilayer production
(Elsevier, 2020-06) Peña, Sara Arana; Rios, Nathalia Saraiva; Sanchez, Carmen Mendez; Lokha, Yuliya; Gonçalves, Luciana Rocha Barros; Lafuente, Roberto Fernández
A strategy to obtain biocatalysts formed by three enzyme layers has been designed using lipases A and B from Candida antarctica (CALA and CALB), the lipases from Rhizomucor miehei (RML) and Thermomyces lanuginosus (TLL), and the artificial chimeric phospholipase Lecitase Ultra (LEU). The enzymes were initially immobilized via interfacial activation on octyl-agarose beads, treated with polyethylenimine (PEI) and a new enzyme layer was immobilized on the octyl-enzyme-PEI composite by ion exchange, producing octyl-enzyme-PEI-enzyme biocatalysts. Except when using LEU, when the two-layer biocatalysts, a large percentage of the PEI-immobilized enzyme was released when a new batch of PEI was added. This was prevented by glutaraldehyde crosslinking. The enzyme modifications produced more active preparations in some cases while in other cases, the effect of the modifications was negative for enzyme activity. These effects of the enzymes modifications were also different when the enzyme was immobilized by interfacial activation or by ion exchange. In all cases, the 3-layer biocatalysts were more active than the single- or bi-layer biocatalysts with some of the assayed substrates. However, as the substrate diffusion problems increased when new enzyme layers were added, even a decrease in enzyme activity with some substrates was found after increasing the number of enzyme layers

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Navegando por Autor "Peña, Sara Arana"