Modifying enzyme activity and selectivity by immobilization

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.

Amination of enzymes to improve biocatalyst performance: coupling genetic modification and physicochemical tools

Improvement of the features of an enzyme is in many instances a pre-requisite for the industrial implementation of these exceedingly interesting biocatalysts. To reach this goal, the researcher may utilize different tools. For example, amination of the enzyme surface produces an alteration of the isoelectric point of the protein along with its chemical reactivity (primary amino groups are the most widely used to obtain the reaction of the enzyme with surfaces, chemical modifiers, etc.) and even its “in vivo” behavior. This review will show some examples of chemical (mainly modifying the carboxylic groups using the carbodiimide route), physical (using polycationic polymers like polyethyleneimine) and genetic amination of the enzyme surface. Special emphasis will be put on cases where the amination is performed to improve subsequent protein modifications. Thus, amination has been used to increase the intensity of the enzyme/support multipoint covalent attachment, to improve the interaction with cation exchanger supports or polymers, or to promote the formation of crosslinkings (both intra-molecular and in the production of crosslinked enzyme aggregates). In other cases, amination has been used to directly modulate the enzyme properties (both in immobilized or free form). Amination of the enzyme surface may also pursue other goals not related to biocatalysis. For example, it has been used to improve the raising of antibodies against different compounds (both increasing the number of haptamers per enzyme and the immunogenicity of the composite) or the ability to penetrate cell membranes. Thus, amination may be a very powerful tool to improve the use of enzymes and proteins in many different areas and a great expansion of its usage may be expected in the near future.

X lberoamerican Conference on Phase Equilibria and Fluid Properties for Process Design (EQUIFASE): June 28-July 1, 2015 - Alicante (Spain): Book of Abstracts

The general purpose of the EQUIFASE Conference is to promote the Scientific and Technologic exchange between people from both the academic and the industrial environment in the field of Phase Equilibria and Thermodynamic Properties for the Design of Chemical Processes. Topics: Measurement of Thermodynamic Properties. Phase Equilibria and Chemical Equilibria. Theory and Modelling. Alternative Solvents. Supercritical Fluids. Ionic Liquids. Energy. Gas and oil. Petrochemicals. Environment and sustainability. Biomolecules and Biotechnology. Product and Process Design. Databases and Software. Education.

Strategies for the one-step immobilization–purification of enzymes as industrial biocatalysts

In this review, we detail the efforts performed to couple the purification and the immobilization of industrial enzymes in a single step. The use of antibodies, the development of specific domains with affinity for some specific supports will be revised. Moreover, we will discuss the use of domains that increase the affinity for standard matrices (ionic exchangers, silicates). We will show how the control of the immobilization conditions may convert some unspecific supports in largely specific ones. The development of tailor-made heterofunctional supports as a tool to immobilize–stabilize–purify some proteins will be discussed in deep, using low concentration of adsorbent groups and a dense layer of groups able to give an intense multipoint covalent attachment. The final coupling of mutagenesis and tailor made supports will be the last part of the review.

Heterofunctional Supports in Enzyme Immobilization: From Traditional Immobilization Protocols to Opportunities in Tuning Enzyme Properties

A heterofunctional support for enzyme immobilization may be defined as that which possesses several distinct functionalities on its surface able to interact with a protein. We will focus on those supports in which a final covalent attachment between the enzyme and the support is achieved. Heterofunctionality sometimes has been featured in very old immobilization techniques, even though in many instances it has been overlooked, giving rise to some misunderstandings. In this respect, glutaraldehyde-activated supports are the oldest multifunctional supports. Their matrix has primary amino groups, the hydrophobic glutaraldehyde chain, and can covalently react with the primary amino groups of the enzyme. Thus, immobilization may start (first event of the immobilization) via different causes and may involve different positions of the enzyme surface depending on the activation degree and immobilization conditions. Other “classical” heterofunctional supports are epoxy commercial supports consisting of reactive covalent epoxy groups on a hydrophobic matrix. Immobilization is performed at high ionic strength to permit protein adsorption, so that covalent attachment may take place at a later stage. Starting from these old immobilization techniques, tailor-made heterofunctional supports have been designed to permit a stricter control of the enzyme immobilization process. The requirement is to find conditions where the main covalent reactive moieties may have very low reactivity toward the enzyme. In this Review we will discuss the suitable properties of the groups able to give the covalent attachment (intending a multipoint covalent attachment), and the groups able to produce the first enzyme adsorption on the support. Prospects, limitations, and likely pathways for the evolution (e.g., coupling of site-directed mutagenesis and thiol heterofunctional supports of enzyme immobilization on heterofunctional supports) will be discussed in this Review.

No tan diferentes. La actividad empresarial de los siderúrgicos españoles en el contexto europeo (1891-1936)

A la hora de explicar las causas del atraso relativo de España en las décadas previas a la Guerra Civil, algunos autores han considerado como un factor clave la mayor propensión de los empresarios españoles a las estrategias de búsqueda de rentas, lo que dio lugar a un sector industrial protegido, cartelizado e ineficiente. Dado que la siderurgia es señalada frecuentemente como el paradigma de las funciones improductivas de los empresarios españoles, el presente trabajo pretende, en primer lugar, contrastar las actitudes de los siderúrgicos españoles con las de los franceses y alemanes en las décadas que precedieron a la Primera Guerra Mundial. En segundo lugar, se analizará si la cartelización del mercado entre 1897 y 1936 tuvo consecuencias negativas para el desarrollo económico de España, tales como restricción a la entrada de competidores, ausencia de innovaciones tecnológicas y, como resultado de ello, una oferta rígida para su producción.

La mediación universitaria: un recurso de orientación. Experiencia en la Universidad de Alicante

Este trabajo de investigación parte del concepto de orientación que compartimos con Bisquerra (2008), definiéndola como un proceso de ayuda continuo a todas las personas, en todos los aspectos, con la finalidad de potenciar el desarrollo humano a lo largo de toda la vida. En este marco de referencia caben todas las acciones dirigidas hacia quien necesita y busca apoyo para su crecimiento personal, académico y profesional. En el ámbito universitario, históricamente poco receptivo a este tipo de actuaciones, se vienen generalizando a raíz de las nuevas concepciones metodológicas que exige la convergencia europea conceptos novedosos e innovadores y que avalan la orientación en la Universidad, como tutorización, transición universitaria, o mediación. Y son ya muchas instituciones las que han puesto en marcha organismos y programas de innovación para su desarrollo efectivo. La Universidad de Alicante viene trabajando en estos temas desde hace unos años y una de las experiencias más novedosas la desarrolla la Facultad de Educación, implantando la figura de la mediadora como recurso de resolución de situaciones conflictivas. En el marco de esta innovación nos planteamos abordar qué se entiende por mediación y cuál es su funcionalidad, al tiempo que realizar una reflexión sobre los agentes implicados y las competencias necesarias para su implementación. Ha participado en la investigación un grupo de alumnas solicitantes del recurso y un equipo docente constituido en Red de investigación. De los resultados obtenidos en la búsqueda de delimitación conceptual y funcional, de estrategias de gestión y de propuestas de actuación en el proceso mediador podemos concluir que es un aspecto positivo y necesario que rentabiliza las tareas de gestión universitaria y que soluciona al alumnado situaciones que pueden entorpecer un desarrollo personal y académico adecuado.