Rafael C. Rodrigues

Bio: Rafael C. Rodrigues is an academic researcher from Universidade Federal do Rio Grande do Sul. The author has contributed to research in topics: Immobilized enzyme & Lipase. The author has an hindex of 44, co-authored 139 publications receiving 9490 citations. Previous affiliations of Rafael C. Rodrigues include Spanish National Research Council & University of Beira Interior.

Modifying enzyme activity and selectivity by immobilization.

Abstract: 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.

Potential of Different Enzyme Immobilization Strategies to Improve Enzyme Performance

Abstract: 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).

Glutaraldehyde in bio-catalysts design: a useful crosslinker and a versatile tool in enzyme immobilization

Abstract: 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.

Importance of the Support Properties for Immobilization or Purification of Enzymes

Abstract: We gratefully recognize the support from the Spanish Government, CTQ2013-41507-R, Colciencia (Colombia) and CNPq (Brazil). The predoctoral fellowships for Mr dos Santos (CNPq, Brazil) are also recognized. A. Berenguer-Murcia thanks the Spanish Ministerio de Ciencia e Innovaciun for a Ramon y Cajal fellowship (RyC-2009-03813).

Enzyme immobilisation in biocatalysis : Why, what and how

Abstract: In this tutorial review, an overview of the why, what and how of enzyme immobilisation for use in biocatalysis is presented. The importance of biocatalysis in the context of green and sustainable chemicals manufacture is discussed and the necessity for immobilisation of enzymes as a key enabling technology for practical and commercial viability is emphasised. The underlying reasons for immobilisation are the need to improve the stability and recyclability of the biocatalyst compared to the free enzyme. The lower risk of product contamination with enzyme residues and low or no allergenicity are further advantages of immobilised enzymes. Methods for immobilisation are divided into three categories: adsorption on a carrier (support), encapsulation in a carrier, and cross-linking (carrier-free). General considerations regarding immobilisation, regardless of the method used, are immobilisation yield, immobilisation efficiency, activity recovery, enzyme loading (wt% in the biocatalyst) and the physical properties, e.g. particle size and density, hydrophobicity and mechanical robustness of the immobilisate, i.e. the immobilised enzyme as a whole (enzyme + support). The choice of immobilisate is also strongly dependent on the reactor configuration used, e.g. stirred tank, fixed bed, fluidised bed, and the mode of downstream processing. Emphasis is placed on relatively recent developments, such as the use of novel supports such as mesoporous silicas, hydrogels, and smart polymers, and cross-linked enzyme aggregates (CLEAs).

Modifying enzyme activity and selectivity by immobilization.

Abstract: 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.

Potential of Different Enzyme Immobilization Strategies to Improve Enzyme Performance

Abstract: 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).

Role of Biocatalysis in Sustainable Chemistry

Abstract: Based on the principles and metrics of green chemistry and sustainable development, biocatalysis is both a green and sustainable technology. This is largely a result of the spectacular advances in molecular biology and biotechnology achieved in the past two decades. Protein engineering has enabled the optimization of existing enzymes and the invention of entirely new biocatalytic reactions that were previously unknown in Nature. It is now eminently feasible to develop enzymatic transformations to fit predefined parameters, resulting in processes that are truly sustainable by design. This approach has successfully been applied, for example, in the industrial synthesis of active pharmaceutical ingredients. In addition to the use of protein engineering, other aspects of biocatalysis engineering, such as substrate, medium, and reactor engineering, can be utilized to improve the efficiency and cost-effectiveness and, hence, the sustainability of biocatalytic reactions. Furthermore, immobilization of an enzyme ...

Industrial use of immobilized enzymes

Abstract: Although many methods for enzyme immobilization have been described in patents and publications, relatively few processes employing immobilized enzymes have been successfully commercialized. The cost of most industrial enzymes is often only a minor component in overall process economics, and in these instances, the additional costs associated with enzyme immobilization are often not justified. More commonly the benefit realized from enzyme immobilization relates to the process advantages that an immobilized catalyst offers, for example, enabling continuous production, improved stability and the absence of the biocatalyst in the product stream. The development and attributes of several established and emerging industrial applications for immobilized enzymes, including high-fructose corn syrup production, pectin hydrolysis, debittering of fruit juices, interesterification of food fats and oils, biodiesel production, and carbon dioxide capture are reviewed herein, highlighting factors that define the advantages of enzyme immobilization.