Immobilization of Proteins in Poly-Styrene-Divinylbenzene Matrices: Functional Properties and Applications

Supports based on poly-styrene-divinylbenzene (PSD) are commercially available since a long time ago. However, they are not commonly used as enzyme immobilization matrices. The main reason for this lies in the negative effect of the very hydrophobic surface on enzyme stability that produces the instantaneous enzyme inactivation in many instances. However, they have recently regained some impact in enzyme immobilization. They are easy to modify, and have been prepared with different modifiers. We will pay special attention to the coating of these supports with ionic liquids, which permits to have the ionic liquid phase anchored to the solid and modulate the enzyme properties without risk of losing these expensive and potentially toxic compounds. Thus, this review will present the covalent or physical immobilization of enzymes on PSD supports, submitted to different modifications. Moreover, lipases immobilized via interfacial activation on some naked PSD supports have shown some unexpected improvement in their catalytic properties, with uses in reactions like hydrolysis, esterification or transesterification.We gratefully recognize the support from the Spanish Government, CTQ2013-41507-R and CNPq (Brazil). The predoctoral fellowships for Ms. García-Galán (Spanish Government), Mr K. Hernandez (I3P-CSIC) and Mr dos Santos (CNPq, Brazil) are also recognized. ). Á. Berenguer-Murcia thanks the Spanish Ministerio de Ciencia e Innovacion for a Ramon y Cajal fellowship (RyC-2009-03813)

Chemical modification of lipase b of candida antarctica in solid phase for improving biochemical properties

Trabajo presentado en el VII Workshop on Biocatalysis and Biotransformations - 1º Simposio Latinoamericano de Biocatalisis y Biotransformaciones, celebrado en Búzios (Brasil) del 23 al 26 de septiembre de 2014.[Introduction]: Chemical modification of enzymes is a strategy widely used in biocatalysis for design of biocatalysts with improved biochemical properties. when the chemical modification is performed on immobilized enzymes, we can protect the enzyme from side-effects that are normally produced with modification of the soluble form of the enzymes. In this work, we modified CALB through two differentmethodologies, and the effect of these modification on the activity, stability and specificity of the enzyme was studied.[Results and discussion]: The effect of chemical modification on the enzyme activity of CALB immobilized on octyl-agarose (OC) and Cyanogen Bromide-agarose (BrCN) were evaluated. In Table 1 are shown changes in enzyme activity of CALB by chemical modification produced by modification (100%) with EDA and TNBS.In Table 2 are shown enzyme activity and enantioselectivity of the different derivatives on (R/S)-Methyl mandelate. Modification of lipase caused different activity and enatioselectivity results, demonstrating that both activity and selectivity of lipase are affected depending on the type of enzyme modification.[Conclusion]: This work exemplifies as chemical modification on solid phase allows modulating biochemical properties of lipases, affecting activity, stability and enantioselectivity of these enzymes.This work was funded by COLCIENCIAS (Project No. 1102-489-25428), Universidad Industrial de Santander (VIE-UIS) and by Ministerio de Ciencia e Innovación from Spain (CTQ2013- 41507-R )..Peer Reviewe

Versatility of glutaraldehyde to immobilize of lipase B from Candida Antarctica- Effect of the immobilization protocol on biocatalytic properties

Trabajo presentado en el VII Workshop on Biocatalysis and Biotransformations - 1º Simposio Latinoamericano de Biocatalisis y Biotransformaciones, celebrado en Búzios (Brasil) del 23 al 26 de septiembre de 2014.[Introduction]: Lipases are enzymes which have a high affinity for hydrophobic surfaces. This is a consequence of the catalytic mechanism of lipases, designed to act in the interface of oil drops, and exhibited the so-called interfacial activation. This work shows how the immobilization of lipases using glutaraldehyde chemistry allows a high versatility, being possible to control the driving force of the immobilization by controlling the experimental conditions: direct covalent attachment using salt and a detergent, ionic exchange using a nonionic detergent and interfacial activation using salt.[Results and discussion]: These different protocols have permitted immobilizing the CALB at different rates, and also to have CALB preparations with quite different properties, mainly when used versus complex substrates. The table 1 shows the specific activities of the different immobilized enzymes, compared to that of the soluble enzyme and the enzyme immobilized in octyl agarose. Considering the sensibility of lipases to the immobilization conditions, differences can be not only attributed to different orientations of the enzyme on the support; the effects of the immobilization medium could also contribute to the observed differences. These differences become very significant studying activity and enantioselectivity of the immobilized enzymes versus R/S methyl mandelate, where the values and their dependence on the experimental conditions are completely different.[Conclusion]: A controlled immobilization of CALB using glutaraldehyde chemistry may contribute to obtaining at least, five different catalysts with very different properties and response to changes in the reaction conditions.This work was supported by grant CTQ2009- 07568 from Spanish Government and VIE-UIS Research Program.Peer Reviewe

Tuning the catalytic properties of lipases immobilized on divinylsulfone activated agarose by altering its nanoenvironment

Lipase from Thermomyces lanuginosus (TLL) and lipase B from Candida antarctica (CALB) have been immobilized on divinylsulfone (DVS) activated agarose beads at pH 10 for 72 h. Then, as a reaction end point, very different nucleophiles have been used to block the support and the effect of the nature of the blocking reagent has been analyzed on the features of the immobilized preparations. The blocking has generally positive effects on enzyme stability in both thermal and organic solvent inactivations. For example, CALB improved 7.5-fold the thermal stability after blocking with imidazole. The effect on enzyme activity was more variable, strongly depending on the substrate and the experimental conditions. Referring to CALB; using p-nitrophenyl butyrate (p-NPB) and methyl phenylacetate, activity always improved by the blocking step, whatever the blocking reagent, while with methyl mandelate or ethyl hexanoate not always the blocking presented a positive effect. Other example is TLL-DVS biocatalyst blocked with Cys. This was more than 8 times more active than the non-blocked preparation and become the most active versus p-NPB at pH 7, the least active versus methyl phenylacetate at pH 5 but the third one most active at pH 9, versus methyl mandelate presented lower activity than the unblocked preparation at pH 5 and versus ethyl hexanoate was the most active at all pH values. That way, enzyme specificity could be strongly altered by this blocking step.We gratefully recognize the support from the Spanish Government, grant CTQ2013-41507-R. The predoctoral fellowships for Mr dos Santos (CNPq, Brazil) and Ms Rueda (Colciencias, Colombia) are also recognized. The authors wish to thank Mr. Ramiro Martínez (Novozymes, Spain) for kindly supplying the enzymes used in this research.Peer Reviewe

Chemical amination of lipases improves their immobilization on octyl-glyoxyl agarose beads

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.We gratefully recognize the support from the MINECO of Spanish Government, CTQ2013-41507-R. The predoctoral fellowships for Ms. Rueda (Colciencias, Colombian Government) and Mr. dos Santos (CNPq, Brazil) are also recognized. The authors wish to thank Mr. Ramiro Martínez (Novozymes, Spain) for kindly supplying the enzymes used in this research.Peer Reviewe

Versatility of divinylsulfone supports permits the tuning of CALB properties during its immobilization

The lipase B from C. antarctica (CALB) has been immobilized on divinylsulfone (DVS) activated agarose beads under different conditions (pH 5–10). In the presence of 0.3% Triton X-100, the immobilization rate was rapid at pH 10 and the slowest one was at pH 5. Incubation at pH 10 for 72 h of the immobilized enzymes before blocking of the support with ethylenediamine permitted improvement of the enzyme stability. Enzyme features (activity, stability, specificity versus different substrates, effect of the pH on enzyme properties) were quite different on the different CALB preparations, suggesting the different orientation of the enzyme. The alkaline incubation produced an increase in enzyme activity with some substrates, and some of the DVS-CALB preparations exhibited a higher specific activity than the octyl-preparations. The indirect fluorescence spectrum of the different immobilized preparations confirmed that different structures of the CALB molecules were generated after immobilization.We gratefully recognize the support from the MINECO of Spanish Government, by the grant CTQ2013-41507-R. The predoctoral fellowships for Ms Rueda (Colciencias, Colombian Goberment) and Mr dos Santos (CNPq, Brazil) are also recognized.Peer Reviewe

Reversible Immobilization of Lipases on Heterofunctional Octyl-Amino Agarose Beads Prevents Enzyme Desorption

Two different heterofunctional octyl-amino supports have been prepared using ethylenediamine and hexylendiamine (OCEDA and OCHDA) and utilized to immobilize five lipases (lipases A (CALA) and B (CALB) from Candida antarctica, lipases from Thermomyces lanuginosus (TLL), from Rhizomucor miehei (RML) and from Candida rugosa (CRL) and the phospholipase Lecitase Ultra (LU). Using pH 5 and 50 mM sodium acetate, the immobilizations proceeded via interfacial activation on the octyl layer, after some ionic bridges were established. These supports did not release enzyme when incubated at Triton X-100 concentrations that released all enzyme molecules from the octyl support. The octyl support produced significant enzyme hyperactivation, except for CALB. However, the activities of the immobilized enzymes were usually slightly higher using the new supports than the octyl ones. Thermal and solvent stabilities of LU and TLL were significantly improved compared to the OC counterparts, while in the other enzymes the stability decreased in most cases (depending on the pH value). As a general rule, OCEDA had lower negative effects on the stability of the immobilized enzymes than OCHDA and while in solvent inactivation the enzyme molecules remained attached to the support using the new supports and were released using monofunctional octyl supports, in thermal inactivations this only occurred in certain cases.We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).We gratefully recognize the support from the MINECO from Spanish Government, (project number CTQ2013-41507-R). The predoctoral fellowships for Rueda (Colciencias, Colombian Government and Becas Iberoamérica “Jóvenes Investigadores”, Banco Santander) and dos Santos and Albuquerque (CNPq, Brazil) are also recognized. The authors wish to thank Ramiro Martínez (Novozymes, Spain) for kindly supplying some of the enzymes used in this research

Reactivation of lipases by the unfolding and refolding of covalently immobilized biocatalysts

Lipases from Candida antarctica (isoform B) (CALB) and Thermomyces lanuginosus (TLL) have been immobilized either covalently or by interfacial activation versus an octyl support, followed by covalent attachment by glyoxyl groups using octyl–glyoxyl agarose beads (OCGLX). These biocatalysts have been submitted to successive cycles of unfolding by incubation in 9 M guanidine and refolding by incubation in aqueous 100 mM phosphate buffer at pH 7, before and after total inactivation in the presence of organic solvents. The four preparations have been reactivated to some extent using this strategy, but the results depended on the method of preparation. Glyoxyl-immobilized CALB may recover 100% of its activity versus p-nitrophenyl butyrate, but after solvent inactivation the recovery of activity was reduced to 95%. The pure covalent TLL preparation recovered around 80% of its activity, either before or after solvent inactivation. Both enzymes showed less recovery of activity using OCGLX, as might be expected from the hydrophobic nature of the supporting groups (60% for CALB and 45% for TLL). In addition, using enzymes previously inactivated by solvent, recovery decreased by 5–10%. These values were maintained through three successive cycles. However, using R- and S-methyl mandelate, it was clear that recovery of activity decreased with further reactivation cycles. Taken as a whole, the unfolding and refolding effect may be used to recover a degree of enzyme activity. This is relevant in terms of application, as it may allow the enzyme preparations to be used for a longer period. However, to reach a similar enzyme structure in each reactivation cycle, it will be necessary to undertake further studies involving the use of other supports in order to improve the unfolding and refolding steps.We are grateful for the support provided by the MINECO of Spanish Government, CTQ2013–41507 R. The predoctoral fellowships for Ms Rueda (Colciencias, Colombian Government) and Mr dos Santos (CNPq, Brazil) are also acknowledged.Peer Reviewe

Improved performance of lipases immobilized on heterofunctional octyl-glyoxyl agarose beads

A new heterofunctional support, octyl-glyoxyl agarose, is proposed in this study. The supports were prepared by simple periodate oxidation of the commercial octyl-agarose, introducing 25 μmol of glyoxyl groups per wet gram of support. This support was assayed with three different lipases (those from Candida antarctica (form B), Thermomyces lanuginosus (TLL) or Rhizomucor miehei) and the artificial phospholipase Lecitase Ultra. Used at pH 7, the new support maintained as first immobilization step the lipase interfacial activation. Thus, it was possible to have the purification and immobilization of the enzyme in one step. Moreover, stabilization of the open form of the lipase was achieved. The covalent enzyme/support bonds cannot be obtained if the immobilized enzyme was not incubated at alkaline pH value. This incubation at pH 10 of the previously immobilized enzymes produced a smaller decrease in enzyme activity when compared to the direct immobilization of the enzymes on glyoxyl-agarose at pH 10, because the immobilization via interfacial activation promoted a stabilization of the lipases. Except in the case of TLL (covalent attachment involved 70% of the enzyme molecules), covalent immobilization yield was over 80%. The non-covalent attached enzyme molecules were discarded by washings with detergent solutions and the new biocatalysts were compared to the octyl-agarose immobilized enzymes. While the stability in thermal and organic solvents inactivations was increased for Lecitase Ultra, CALB and RML, TLL improved its stability in organic media but its thermal stability decreased after covalent attachment of the interfacially activated enzyme. This stabilization resulted in octyl-glyoxyl-lipase preparations which presented higher activity in the presence of organic solvents. Finally, while octyl-agarose released enzyme molecules after incubation at high temperatures or in the presence of organic solvents and detergents, the covalently immobilized enzyme remained attached to the support even after boiling the enzyme in SDS, eliminating the risks of product contamination.We gratefully recognize the support from the MINECO of Spanish Government, CTQ2013-41507-R. The predoctoral fellowships for Ms. Rueda (Colciencia, Colombian Goberment) and Mr dos Santos (CNPq, Brazil) are also recognized.Peer Reviewe

Bovine trypsin immobilization on agarose activated with divinylsulfone: Improved activity and stability via multipoint covalent attachment

© 2015 Elsevier B.V. All rights reserved. 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.We gratefully recognize the support from the Spanish Government (MINECO; grant CTQ2013-41507-R). The predoctoral fellowships for Mr dos Santos (CNPq, Brazil) and Ms Rueda (Colciencias, Colombia) are also recognized. The help and comments from Dr. Ángel Berenguer (Instituto de Materiales, Universidad de Alicante) are kindly acknowledgedPeer Reviewe