ICP CSIC Campus UAM CSIC

Madrid, Spain

ICP CSIC Campus UAM CSIC

Madrid, Spain
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Manoel E.A.,Federal University of Rio de Janeiro | Pinto M.,Federal University of Rio de Janeiro | Dos Santos J.C.S.,ICP CSIC Campus UAM CSIC | Dos Santos J.C.S.,University da Integracao Internacional da Lusofonia Afro Brasileira | And 5 more authors.
RSC Advances | Year: 2016

Different core-shell polymeric supports, exhibiting different morphologies and composition, were produced through simultaneous suspension and emulsion polymerization, using styrene (S) and divinylbenzene (DVB) as co-monomers. Supports composed of polystyrene in both the core and the shell (PS/PS) and the new poly(styrene-co-divinylbenzene) support (PS-co-DVB/PS-co-DVB) were used for the immobilization of three different lipases (from Rhizomucor miehie (RML), from Themomyces lanuginosus (TLL) and the form B from Candida antarctica, (CALB)) and of the phospholipase Lecitase Ultra (LU). The features of the new biocatalysts were evaluated and compared to the properties of commercial biocatalysts (Novozym 435 (CALB), Lipozyme RM IM and Lipozyme TL IM) and biocatalysts prepared by enzyme immobilization onto commercial octyl-agarose, a support reported as very suitable for lipase immobilization. It was shown that protein loading and stability of the biocatalysts prepared with the core-shell supports were higher than the ones obtained with commercial octyl-agarose or the commercial lipase preparations. Besides, it was shown that the biocatalysts prepared with the core-shell supports also presented higher activities than commercial biocatalysts when employing different substrates, encouraging the use of the produced core-shell supports for immobilization of lipases and the development of new applications. © 2016 The Royal Society of Chemistry.


Barbosa O.,Industrial University of Santander | Ortiz C.,Industrial University of Santander | Berenguer-Murcia A.,University of Alicante | Torres R.,Industrial University of Santander | And 2 more authors.
RSC Advances | Year: 2014

Glutaraldehyde is one of the most widely used reagents in the design of biocatalysts. It is a powerful crosslinker, able to react with itself, with the advantages that this may bring forth. In this review, we intend to give a general vision of its potential and the precautions that must be taken when using this effective reagent. First, the chemistry of the glutaraldehyde/amino reaction will be commented upon. This reaction is still not fully clarified, but it seems to be based on the formation of 6-membered heterocycles formed by 5 C and one O. Then, we will discuss the production of intra- and inter-molecular enzyme crosslinks (increasing enzyme rigidity or preventing subunit dissociation in multimeric enzymes). Special emphasis will be placed on the preparation of cross-linked enzyme aggregates (CLEAs), mainly in enzymes that have low density of surface reactive groups and, therefore, may be problematic to obtain a final solid catalyst. Next, we will comment on the uses of glutaraldehyde in enzymes previously immobilized on supports. First, the treatment of enzymes immobilized on supports that cannot react with glutaraldehyde (only inter and intramolecular cross-linkings will be possible) to prevent enzyme leakage and obtain some enzyme stabilization via cross-linking. Second, the cross-linking of enzymes adsorbed on aminated supports, where together with other reactions enzyme/support crosslinking is also possible; the enzyme is incorporated into the support. Finally, we will present the use of aminated supports preactivated with glutaraldehyde. Optimal glutaraldehyde modifications will be discussed in each specific case (one or two glutaraldehyde molecules for amino group in the support and/or the protein). Using preactivated supports, the heterofunctional nature of the supports will be highlighted, with the drawbacks and advantages that the heterofunctionality may have. Particular attention will be paid to the control of the first event that causes the immobilization depending on the experimental conditions to alter the enzyme orientation regarding the support surface. Thus, glutaraldehyde, an apparently old fashioned reactive, remains the most widely used and with broadest application possibilities among the compounds used for the design of biocatalyst. © 2014 The Royal Society of Chemistry.


Rueda N.,ICP CSIC Campus UAM CSIC | Rueda N.,Industrial University of Santander | Santos J.C.S.D.,ICP CSIC Campus UAM CSIC | Santos J.C.S.D.,Federal University of Ceará | And 4 more authors.
Catalysis Today | Year: 2015

This paper describes a new strategy that permits to take full advantage of octyl-agarose supports to immobilize lipases (one-step purification and immobilization, stabilization of the open form of the enzyme) but that may be used in any reaction media. To this purpose, we have utilized aminated lipases and glyoxyl-octyl agarose (OCGLX). As model enzymes, we have used lipase B from Candida antarctica, lipase from Thermomyces lanuginosus and lipase from Rhizomucor miehei (RML). The amination of the enzyme may be performed in the enzymes already adsorbed on OCGLX, greatly simplifying the protocol. The immobilization was carried out at pH 5 to ensure the immobilization via interfacial activation versus the hydrophobic support, and afterwards the pH was increased to pH 9 or 10 to promote some covalent attachments. 100% of the aminated lipases became covalently immobilized on OCGLX after 2. h even at pH 9, while using unmodified enzymes some enzyme molecules could be desorbed from the support even after 24. h of incubation at pH 10, with a significantly lower cost in terms of activity. The resulting biocatalysts have a significant improved stability compared to the non-aminated OCGLX preparations. Amination in some instances presented positive effects on enzyme properties, while in other cases the effects were negative. However, the covalent immobilization at OCGLX compensated the negative effects and increases the positive ones. In some cases, the stabilization factor become 40-50 when compared with the use of non-aminated enzyme (e.g., using RML), and retained a high percentage of hydrolytic activity in the presence of acetonitrile concentration as high as 90%, where the enzyme immobilized on octyl supports could be desorbed. © 2015 Elsevier B.V.


Dal Magro L.,Federal University of Rio Grande do Sul | Hertz P.F.,Federal University of Rio Grande do Sul | Fernandez-Lafuente R.,ICP CSIC Campus UAM CSIC | Klein M.P.,Federal University of Rio Grande do Sul | And 2 more authors.
RSC Advances | Year: 2016

Combined cross-linked enzyme aggregates (combi-CLEAs) are a novel prospect for immobilization of a mixture of enzymes and the present study addresses the preparation, characterization and application of pectinases-cellulases combi-CLEAs for grape juice clarification. Initially, 8 enzymatic preparations were tested for turbidity reduction in grape juice and Rohapect 10L provided the best results (around 50% in 1 h) being selected for CLEA preparation. The optimization of combi-CLEAs, was performed using a central composite design (CCD) and response surface methodology (RSM) varying the glutaraldehyde concentration and reaction time, using isopropanol as the precipitant agent. The best conditions for the Rohapect 10L CLEA preparation was 110 mM of glutaraldehyde and 2 h. Bovine serum albumin (BSA) was used as a feeder and improved the volumetric activity, recovered activity and thermal stability. Combi-CLEAs-BSA prepared using 0.4 mg mL-1 of the enzyme mixture and 2.4 mg mL-1 of BSA presented an activity of 14 U mL-1, 18% of recovered activity and 3-times more thermal stability compared to soluble enzymes. The combi-CLEAs and combi-CLEAs-BSA were tested in repeated batches, being reused for 4 and 6 cycles, respectively, keeping 100% of the initial activity. The combi-CLEAs and combi-CLEAs-BSA appear to be suitable alternatives of immobilized biocatalyst for the clarification of grape juices. © 2016 The Royal Society of Chemistry.


Dos Santos J.C.S.,ICP CSIC Campus UAM CSIC | Dos Santos J.C.S.,Federal University of Ceará | Rueda N.,ICP CSIC Campus UAM CSIC | Rueda N.,Industrial University of Santander | And 6 more authors.
Journal of Molecular Catalysis B: Enzymatic | Year: 2015

Trypsin has been immobilized on divinyl sulfone (DVS) activated agarose at pH 5, 7 and 10. While at pH 5 and 7 immobilization was slow and presented a negative effect on enzyme activity, the immobilization at pH 10 produced a significant increment of activity (by a 24 fold factor). Using this preparation, the effect on enzyme activity/stability of different blocking reagents (used as an enzyme-support reaction end point) were evaluated, selecting ethylenediamine (EDA) because it produced an increase in enzyme activity (a 4 fold factor) and the best results in terms of stability. Next, the effect of alkaline incubation on enzyme activity/stability before the blocking step was analyzed. Activity decreased by 40% after 72 h (but it should be considered that previously it had increased by a 24 fold factor), but the stability significantly improved after this incubation. Thus, after immobilization at different pH values, the immobilized trypsin was submitted to 72 h of alkaline incubation and blocked with EDA. The most active and stable preparation was that immobilized at pH 10. This preparation was less stable than the glyoxyl preparation in thermal inactivations (by less than a twofold factor), but was more stable in organic solvent inactivation (also by less than a twofold factor). The number of groups involved in the enzyme support attachment was 6 Lys using glyoxyl and became a minimum of 13 (including Lys, Tyr and His) using the DVS-activated support (the precision of the method did not permit to analyze the implication of some of the 3 terminal amino groups). Thus, this DVS-agarose support seems to be a very promising support to permit a very intense enzyme-support multipoint covalent attachment. © 2015 Elsevier B.V. All rights reserved.


Suescun A.,Industrial University of Santander | Rueda N.,Industrial University of Santander | Dos Santos J.C.S.,ICP CSIC Campus UAM CSIC | Dos Santos J.C.S.,Federal University of Ceará | And 5 more authors.
Process Biochemistry | Year: 2015

Lipases from Candida rugosa (CRL) and from Candida antarctica (isoform A) (CALA) have been successfully immobilized on octyl-glyoxyl agarose (OCGLX) beads and compared to the octyl-agarose (OC) or glyoxyl (GLX) beads immobilized counterparts. Immobilization on OCGLX gave similar hyperactivations than those found for the immobilization on OC supports, although the incubation at pH 10.0 for 4 h decreased the activity of both enzymes by 25%. After reduction, more than 95% of the enzyme activity was covalently attached to the support. The fraction not covalently attached was desorbed by washing with detergent. These biocatalysts were more stable than the octyl counterparts in thermal or organic solvent inactivation. More interestingly, the irreversible immobilization permitted the reactivation of CALA biocatalysts inactivated by incubation in organic solvent, after unfolding in the presence of guanidine and refolding in aqueous buffer (around 55% of the activity could be recovered during 3 successive cycles of inactivation/reactivation). GLX-CALA permitted to recover 75% of the activity, but the thermal stability and activity was much lower, and this strategy could not be applied to CRL. Neither the enzyme immobilized on cyanogen bromide nor the enzyme immobilized on OCGLX exhibited significant activity after the unfolding/refolding strategy. © 2015 Elsevier Ltd. All rights reserved.

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