Navegando por Autor "Carballares, Diego"
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Artigo 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 FernandezThis 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 distributionArtigo 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 FernandezLipases 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 resultsArtigo 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 FernandezA 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