Institute Catalisis CSIC

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Institute Catalisis CSIC

Madrid, Spain
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Rodrigues R.C.,Federal University of Rio Grande do Sul | Berenguer-Murcia A.,University of Alicante | Fernandez-Lafuente R.,Institute Catalisis CSIC
Advanced Synthesis and Catalysis | Year: 2011

Chemical modification and immobilization of enzymes have been usually considered unrelated tools to improve biocatalyst features. However, there are many examples where a chemically modified enzyme is finally used in an immobilized form, and that exemplifies how both tools may be complementary resulting in a synergism in the final results. In this review we present some of the strategies that may give that result. For example, the chemical modification of soluble enzymes may be used to improve their immobilization (reinforcing adsorption or improving multipoint covalent attachment), or just to improve enzyme stability and facilitate the selection of the immobilization conditions. Chemical modification of previously immobilized enzymes benefits from solid-phase chemistry due to the nature of enzymes (e.g., prevention of inactivation, aggregation, etc.). The use of different targets for chemical modifications with small molecules or multifunctional polymers are also discussed: intramolecular or intersubunit cross-linking, one-point modification, generation of artificial microenvironments, etc. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Garcia-Galan C.,Institute Catalisis CSIC | Berenguer-Murcia A.,University of Alicante | Fernandez-Lafuente R.,Institute Catalisis CSIC | Rodrigues R.C.,Federal University of Rio Grande do Sul
Advanced Synthesis and Catalysis | Year: 2011

Enzyme biocatalysis plays a very relevant role in the development of many chemical industries, e.g., energy, food or fine chemistry. To achieve this goal, enzyme immobilization is a usual pre-requisite as a solution to get reusable biocatalysts and thus decrease the price of this relatively expensive compound. However, a proper immobilization technique may permit far more than to get a reusable enzyme; it may be used to improve enzyme performance by improving some enzyme limitations: enzyme purity, stability (including the possibility of enzyme reactivation), activity, specificity, selectivity, or inhibitions. Among the diverse immobilization techniques, the use of pre-existing supports to immobilize enzymes (via covalent or physical coupling) and the immobilization without supports [enzyme crosslinked aggregates (CLEAs) or crystals (CLECs)] are the most used or promising ones. This paper intends to give the advantages and disadvantages of the different existing immobilization strategies to solve the different aforementioned enzyme limitations. Moreover, the use of nanoparticles as immobilization supports is achieving an increasing importance, as the nanoparticles versatility increases and becomes more accessible to the researchers. We will also discuss here some of the advantages and drawbacks of these non porous supports compared to conventional porous supports. Although there are no universal optimal solutions for all cases, we will try to give some advice to select the optimal strategy for each particular enzyme and process, considering the enzyme properties, nature of the process and of the substrate. In some occasions the selection will be compulsory, for example due to the nature of the substrate. In other cases the optimal biocatalyst may depend on the company requirements (e.g., volumetric activity, enzyme stability, etc). Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Rodrigues R.C.,Federal University of Rio Grande do Sul | Ortiz C.,Industrial University of Santander | Berenguer-Murcia A.,University of Alicante | Torres R.,Industrial University of Santander | Fernandez-Lafuente R.,Institute Catalisis CSIC
Chemical Society Reviews | Year: 2013

Immobilization of enzymes may produce alterations in their observed activity, specificity or selectivity. Although in many cases an impoverishment of the enzyme properties is observed upon immobilization (caused by the distortion of the enzyme due to the interaction with the support) in some instances such properties may be enhanced by this immobilization. These alterations in enzyme properties are sometimes associated with changes in the enzyme structure. Occasionally, these variations will be positive. For example, they may be related to the stabilization of a hyperactivated form of the enzyme, like in the case of lipases immobilized on hydrophobic supports via interfacial activation. In some other instances, these improvements will be just a consequence of random modifications in the enzyme properties that in some reactions will be positive while in others may be negative. For this reason, the preparation of a library of biocatalysts as broad as possible may be a key turning point to find an immobilized biocatalyst with improved properties when compared to the free enzyme. Immobilized enzymes will be dispersed on the support surface and aggregation will no longer be possible, while the free enzyme may suffer aggregation, which greatly decreases enzyme activity. Moreover, enzyme rigidification may lead to preservation of the enzyme properties under drastic conditions in which the enzyme tends to become distorted thus decreasing its activity. Furthermore, immobilization of enzymes on a support, mainly on a porous support, may in many cases also have a positive impact on the observed enzyme behavior, not really related to structural changes. For example, the promotion of diffusional problems (e.g., pH gradients, substrate or product gradients), partition (towards or away from the enzyme environment, for substrate or products), or the blocking of some areas (e.g., reducing inhibitions) may greatly improve enzyme performance. Thus, in this tutorial review, we will try to list and explain some of the main reasons that may produce an improvement in enzyme activity, specificity or selectivity, either real or apparent, due to immobilization. © 2013 The Royal Society of Chemistry.

Hernandez K.,Institute Catalisis CSIC | Fernandez-Lafuente R.,Institute Catalisis CSIC
Enzyme and Microbial Technology | Year: 2011

Mutagenesis and immobilization are usually considered to be unrelated techniques with potential applications to improve protein properties. However, there are several reports showing that the use of site-directed mutagenesis to improve enzyme properties directly, but also how enzymes are immobilized on a support, can be a powerful tool to improve the properties of immobilized biomolecules for use as biosensors or biocatalysts. Standard immobilizations are not fully random processes, but the protein orientation may be difficult to alter. Initially, most efforts using this idea were addressed towards controlling the orientation of the enzyme on the immobilization support, in many cases to facilitate electron transfer from the support to the enzyme in redox biosensors. Usually, Cys residues are used to directly immobilize the protein on a support that contains disulfide groups or that is made from gold. There are also some examples using His in the target areas of the protein and using supports modified with immobilized metal chelates and other tags (e.g., using immobilized antibodies). Furthermore, site-directed mutagenesis to control immobilization is useful for improving the activity, the stability and even the selectivity of the immobilized protein, for example, via site-directed rigidification of selected areas of the protein. Initially, only Cys and disulfide supports were employed, but other supports with higher potential to give multipoint covalent attachment are being employed (e.g., glyoxyl or epoxy-disulfide supports). The advances in support design and the deeper knowledge of the mechanisms of enzyme-support interactions have permitted exploration of the possibilities of the coupled use of site-directed mutagenesis and immobilization in a new way. This paper intends to review some of the advances and possibilities that these coupled strategies permit. © 2010 Elsevier Inc.

This review article highlights the strategies to successfully perform an efficient solid-phase synthesis of complex peptides including posttranslational modifications, fluorescent labels, and reporters or linking groups of exceptional value for biological studies of several important diseases. The solid-phase approach is the best alternative to synthesize these peptides rapidly and in high amounts. The key aspects that need to be considered when performing a peptide synthesis in solid phase of these molecules are discussed. This journal is © the Partner Organisations 2014.

Fernandez-Lafuente R.,Institute Catalisis CSIC
Journal of Molecular Catalysis B: Enzymatic | Year: 2010

The lipase from Thermomyces laguginosus (formerly Humicola laguginosa) (TLL) is a basophilic and noticeably thermostable enzyme, commercially available in both soluble and immobilized form. Although initially oriented toward the food industry, the enzyme has found applications in many different industrial areas, from biodiesel production to fine chemicals (mainly in enantio and regioselective or specific processes). This review intends to show some of the most relevant aspects of the use of this interesting enzyme. After checking the enzyme features, some of the most efficient methods of TLL immobilization will be commented. Finally, the main uses of the enzyme will be revised, with special emphasis in the modification of fats and oils, production of biodiesel, resolution of racemic mixtures, enantioselective hydrolysis of prochiral esters and regioselective process involving sugar preparations. In many instances, TLL has been compared to other lipases, the advantages or disadvantages of the enzyme will be discussed. © 2009 Elsevier B.V.

Palomo J.M.,Institute Catalisis CSIC | Filice M.,Institute Catalisis CSIC
Biotechnology Advances | Year: 2015

The development of new and successful biotransformation processes of key interest in medicinal and pharmaceutical chemistry involves creating new biocatalysts with improved or even new activities and selectivities.This review emphasizes the new emerging developed strategies to achieve this goal, site-selective chemical modification of enzymes using tailor-made peptides, specific insertion of metals or organometallic complexes into proteins producing bio-catalysts with multiple activities and computational design for creating evolved artificial enzymes with non-natural synthetic catalytic activities. © 2014 Elsevier Inc.

Palomo J.M.,Institute Catalisis CSIC
European Journal of Organic Chemistry | Year: 2010

The Diels-Alder reaction is probably, together with aldol condensation, the most commonly used reaction in chemistry. The Diels-Alder approach, which involves a diene and a dienophile not present in any biomolecule, allows a chemoselective reaction without the need of protecting groups. Moreover, water has an extraordinary rate-accelerating effect on the reaction process. Thus, this chemical approach has been recognized as a promising procedure for protein bioconjugation. In this review some of the most recent advances in the application of the Diels-Alder reaction in selective surface modification, immobilization and biocatalysis will be discussed. The Diels-Alder reaction is one of the most powerful methods for C-C bond formation. This Microreview discusses last recent advances in the application of this cycloaddition on protein modification, immobilization and biocatalysis. Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Palomo J.M.,Institute Catalisis CSIC
Organic and Biomolecular Chemistry | Year: 2012

Click-chemistry is an approach based on cycloaddition reactions which has been successfully used as a chemical approach for complex organic molecules and which has recently starred in a boom in the world of protein chemistry. The advantage of the use of this technique in protein chemistry is based on a very high and efficient chemoselectivity, which usually requires simple or no purification and is extremely rate-accelerated in aqueous media. The perspective discusses some of the most recent advances in the application of this reaction in selective enzyme surface modification for the creation of new semisynthetic enzymes (fluorescence labeled enzymes, peptide-enzyme conjugates, glycosylated enzymes), and interestingly, the recent design and creation of "click" enzymes. © 2012 The Royal Society of Chemistry.

Filice M.,Institute Catalisis CSIC | Palomo J.M.,Institute Catalisis CSIC
ACS Catalysis | Year: 2014

Cascade reactions are an emerging technology in organic chemistry, introducing elegance and efficiency to synthetic strategies. This Review provides an overview of the novel and recent achievements in cascade processes catalyzed by bionanostructures. The examples here selected demonstrate the advances related to the application of heterogeneous nanocatalysts- nanostructures and biomolecules combined by different manner-in efficient cascade processes. Metallic nanoparticles supported on biomolecules, multienzymatic systems or bionanohybrid structures with multicatalytic activities (containing both organometallic and biocatalytic activity) were selectively and efficiently used alone or in cooperative fashion. This Review highlights examples of efficient and interesting catalytic cascade processes in organic chemistry, ultrasensitive biosensing, or energy storage and conversion, underscoring their tremendous future potential in chemical synthesis. © 2014 American Chemical Society.

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