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Biocatalysis is a key technology enabling plastic recycling. However, despite advances done in the development of plastic-degrading enzymes, the molecular mechanisms that govern their catalytic performance are poorly understood, hampering the engineering of more efficient enzyme-based technologies. In this work, we study the hydrolysis of PET-derived diesters and PET trimers catalyzed by the highly promiscuous lipase B from Candida antarctica (CALB) through QM/MM molecular dynamics simulations supported by experimental Michaelis–Menten kinetics. The computational studies reveal the role of the pH on the CALB regioselectivity toward the hydrolysis of bis-(hydroxyethyl) terephthalate (BHET). We exploit this insight to perform a pH-controlled biotransformation that selectively hydrolyzes BHET to either its corresponding diacid or monoesters using both soluble and immobilized CALB. The discoveries presented here can be exploited for the valorization of BHET resulting from the organocatalytic depolymerization of PET.

Immobilized multienzyme systems are gaining momentum in applied biocatalysis; however, the coimmobilization of several enzymes on one carrier is still challenging. In this work, we exploited a heterofunctional support activated with three different chemical functionalities to immobilize a wide variety of different enzymes. This support is based on agarose microbeads activated with aldehyde, amino, and cobalt chelate moieties that allow a fast and irreversible immobilization of enzymes, enhancing the thermostability of most of the heterogeneous biocatalysts (up to 21-fold higher than the soluble one). Furthermore, this trifunctional support serves to efficiently coimmobilize a multienzyme system composed of an alcohol dehydrogenase, a reduced nicotinamide adenine dinucleotide (NADH) oxidase, and a catalase. The confined multienzymatic system demonstrates higher performance than its free counterpart, achieving a total turnover number (TTN) of 1 × 105 during five batch consecutive cycles. We envision this solid material as a platform for coimmobilizing multienzyme systems with enhanced properties to catalyze stepwise biotransformations.

Multi-enzymatic cascades with enzymes arranged in close-proximity through a protein scaffold can trigger a substrate channeling effect, allowing for efficient cofactor reuse with industrial potential. However, precise nanometric organization of enzymes challenges the design of scaffolds. In this study, we create a nanometrically organized multi-enzymatic system exploiting engineered Tetrapeptide Repeat Affinity Proteins (TRAPs) as scaffolding for biocatalysis. We genetically fuse TRAP domains and program them to selectively and orthogonally recognize peptide-tags fused to enzymes, which upon binding form spatially organized metabolomes. In addition, the scaffold encodes binding sites to selectively and reversibly sequester reaction intermediates like cofactors via electrostatic interactions, increasing their local concentration and, consequently, the catalytic efficiency. This concept is demonstrated for the biosynthesis of amino acids and amines using up to three enzymes. Scaffolded multi-enzyme systems present up to 5-fold higher specific productivity than the non-scaffolded ones. In-depth analysis suggests that channeling of NADH cofactor between the assembled enzymes enhances the overall cascade throughput and the product yield. Moreover, we immobilize this biomolecular scaffold on solid supports, creating reusable heterogeneous multi-functional biocatalysts for consecutive operational batch cycles. Our results demonstrate the potential of TRAP-scaffolding systems as spatial-organizing tools to increase the efficiency of cell-free biosynthetic pathways.

Supporting schemes including the preparation of AG-Co2+/A/G, AG-Co2+/A/E, and AG-Co2+/H; supporting figures including the distribution of agarose-activated microbeads with different functionalities, immobilization and thermal inactivation kinetics, enzyme desorption assays, confocal fluorescence microscopy images of AG-Co2+/A/G, spectra of intrinsic protein fluorescence, residual activity of HBs under operation conditions and consumed 1,5-pentanediol after 24 h by the soluble or coimmobilized enzymes; supporting tables including the activation degree of agarose microbeads, heterofunctional activation of agarose microbeads, abbreviation of the differently prepared supports, single-enzyme immobilization parameters on AG-Co2+/E microbeads, and thermal stability of soluble enzymes and individual residual activity of coimmobilized enzymes after five batch cycles.

Production of enantiomerically pure molecules is of utmost importance in the pharma industry. In this context, biocatalysis emerges as an alternative to conventional chemical methods due to the exquisite selectivity and specificity underlying the enzymes. In this work, we design a multienzymatic system to perform the deracemization of alkyl glyceryl ethers as potential building blocks for the synthesis of drugs. The key to success in this route is controlling the spatial organization of the enzymes involved in the cascade through their immobilization on porous carriers. By fine tuning the intraparticle organization of an enzymatic cascade comprising an (S)-selective glycerol dehydrogenase from Bacillus stearothermophilus and an (R)-selective ketoreductase from Lactobacillus kefir, we performed the oxidoreductive deracemization of rac-alkyl/aryl glyceryl ethers with a yield up to 100% and enantiomeric excess e.e. > 99%, otherwise impossible using a soluble system. Remarkably, we find that optimal spatial assembly of the biocatalyst ameliorates the inhibition phenomena experimented by the system and increases the deracemization rate by 4-fold. Finally, integrating an enzymatic nicotinamide adenine dinucleotide oxidized disodium salt (NAD+) regeneration system to the heterogeneous biocatalyst, we intensified the process by reusing it in discontinuous and consecutive batch cycles and scaling the reaction up to 250 mM substrate, achieving 100% yield and e.e. > 99%.

Self-sufficient heterogeneous biocatalysts (ssHB) are promising candidates for implementing cofactor-dependent enzymes in chemical biomanufacturing. Most strategies for coimmobilizing cofactors with dehydrogenases on porous agarose microbeads involve the use of cationic polymers (i.e., polyethylenimine, PEI) that interact electrostatically with the phosphate groups of their corresponding phosphorylated cofactors. Although the latter is a powerful and versatile approach, ionic bonds are disrupted in biotransformations operating at a high ionic strength, where screening of bonded ions takes place. Harnessing the ribose groups present in adenylated cofactors, we immobilize a selection of these (NAD(P)H, NAD(P)+, FAD, and ATP) on agarose microbeads functionalized with boronic acid to establish reversible covalent bonds between the cis-diol of the ribose in the cofactor backbone and the boronic acid. To do so, we functionalize cobalt-activated porous agarose beads with boronic acid (AG-B/Co2+) for the coimmobilization of adenylated cofactors with the corresponding cofactor dependent His-tagged dehydrogenases. First, we demonstrate that the adenylated cofactor-support interactions are reversible but show resistance against high salt concentrations, overcoming the main limitation of the current self-sufficient heterogeneous biocatalysts. Then, we coimmobilized several His-tagged enzymes with their corresponding adenylated cofactors and investigated the functionality and stability of these ssHBs in reductive aminations performed under high ionic strength in both batch and flow reactors. As a result, we manage to reuse the immobilized enzymes and the cofactors 3.5 × 105 and 167 times, respectively. This work expands the usefulness of ssBHs for hitherto bioprocess regardless of the ionic strength of the media.

The oligosaccharides present in milk have health benefits for consumers beyond the expected nutritional value alone. These oligosaccharides can be found free or conjugated to more complex molecules, such as proteins and they not only serve as growth substrate for the intestinal bacteria, they can directly modulate the response of the intestinal cells. Although, their potential benefits on human health are recognized the amount of studies that show such functionality is limited. In the research presented in this thesis, we studied how the oligosaccharide composition affects the immunomodulatory capacity of bovine lactoferrin (bLF). We show that depending on the change of composition of these oligosaccharides the release of molecules that mediate inflammatory pathways via immune sensors called toll-like receptors can be increased or decreased. Our results indicated that also the oligosaccharides removed from bLF have the ability to block inflammatory responses as efficiently as compounds that are clinically used for their anti-inflammatory properties, such as chloroquine. The lactose found in cow milk can be elongated into oligosaccharides that mimic the chemical structures of the oligosaccharides present naturally in human milk. These resulting structures are currently used to fortify infant formula. Molecules such as galacto-oligosaccharides (GOS) and 2’-fucosyllactose (2’-FL) can protect the intestinal barrier against damage. Thanks to recent developments in production and characterization of carbohydrates, it is possible to better understand how oligosaccharides play a role in human health and disease.

[EN] Catalysis-based approaches for the activation of anticancer agents hold considerable promise. These principally rely on the use of metal catalysts capable of deprotecting inactive precursors of organic drugs or transforming key biomolecules available in the cellular environment. Nevertheless, the efficiency of most of the schemes described so far is rather low, limiting the benefits of catalytic amplification as strategy for controlling the therapeutic effects of anticancer compounds. In the work presented here, we show that flavin reactivity within a hydrogel matrix provides a viable solution for the efficient catalytic activation and delivery of cisplatin, a worldwide clinically-approved inorganic chemotherapy agent. This is achieved by ionically adsorbing a flavin catalyst and a Pt(iv) prodrug as substrate into porous amino-functionalized agarose beads. The hydrogel chassis supplies high local concentrations of electron donating groups/molecules in the surrounding of the catalyst, ultimately boosting substrate conversion rates (TOF >200 min(-1)) and enabling controlled liberation of the drug by light or chemical stimuli. Overall, this approach can afford platforms for the efficient delivery of platinum drugs as demonstrated herein by using a transdermal diffusion model simulating the human skin.

[EN] Catalysis-based approaches for the activation of anticancer agents hold considerable promise. These principally rely on the use of metal catalysts capable of deprotecting inactive precursors of organic drugs or transforming key biomolecules available in the cellular environment. Nevertheless, the efficiency of most of the schemes described so far is rather low, limiting the benefits of catalytic amplification as strategy for controlling the therapeutic effects of anticancer compounds. In the work presented here, we show that flavin reactivity within a hydrogel matrix provides a viable solution for the efficient catalytic activation and delivery of cisplatin, a worldwide clinically-approved inorganic chemotherapy agent. This is achieved by ionically adsorbing a flavin catalyst and a Pt(iv) prodrug as substrate into porous amino-functionalized agarose beads. The hydrogel chassis supplies high local concentrations of electron donating groups/molecules in the surrounding of the catalyst, ultimately boosting substrate conversion rates (TOF >200 min(-1)) and enabling controlled liberation of the drug by light or chemical stimuli. Overall, this approach can afford platforms for the efficient delivery of platinum drugs as demonstrated herein by using a transdermal diffusion model simulating the human skin.

[EN] Catalysis-based approaches for the activation of anticancer agents hold considerable promise. These principally rely on the use of metal catalysts capable of deprotecting inactive precursors of organic drugs or transforming key biomolecules available in the cellular environment. Nevertheless, the efficiency of most of the schemes described so far is rather low, limiting the benefits of catalytic amplification as strategy for controlling the therapeutic effects of anticancer compounds. In the work presented here, we show that flavin reactivity within a hydrogel matrix provides a viable solution for the efficient catalytic activation and delivery of cisplatin, a worldwide clinically-approved inorganic chemotherapy agent. This is achieved by ionically adsorbing a flavin catalyst and a Pt(iv) prodrug as substrate into porous amino-functionalized agarose beads. The hydrogel chassis supplies high local concentrations of electron donating groups/molecules in the surrounding of the catalyst, ultimately boosting substrate conversion rates (TOF >200 min(-1)) and enabling controlled liberation of the drug by light or chemical stimuli. Overall, this approach can afford platforms for the efficient delivery of platinum drugs as demonstrated herein by using a transdermal diffusion model simulating the human skin.

The oligosaccharides present in milk have health benefits for consumers beyond the expected nutritional value alone. These oligosaccharides can be found free or conjugated to more complex molecules, such as proteins and they not only serve as growth substrate for the intestinal bacteria, they can directly modulate the response of the intestinal cells. Although, their potential benefits on human health are recognized the amount of studies that show such functionality is limited. In the research presented in this thesis, we studied how the oligosaccharide composition affects the immunomodulatory capacity of bovine lactoferrin (bLF). We show that depending on the change of composition of these oligosaccharides the release of molecules that mediate inflammatory pathways via immune sensors called toll-like receptors can be increased or decreased. Our results indicated that also the oligosaccharides removed from bLF have the ability to block inflammatory responses as efficiently as compounds that are clinically used for their anti-inflammatory properties, such as chloroquine. The lactose found in cow milk can be elongated into oligosaccharides that mimic the chemical structures of the oligosaccharides present naturally in human milk. These resulting structures are currently used to fortify infant formula. Molecules such as galacto-oligosaccharides (GOS) and 2’-fucosyllactose (2’-FL) can protect the intestinal barrier against damage. Thanks to recent developments in production and characterization of carbohydrates, it is possible to better understand how oligosaccharides play a role in human health and disease.

Biocatalytic cascades afford the development of economically sustainable and green processes. Herein we examined the unprecedented coupling of co-immobilised Gluconobacter oxydans and an isolated transaminase to synthesise serinol from glycerol. Through this approach, we manufactured up to 36 mM serinol, the highest titer ever reported for a non-fermentative biosynthesis. More importantly, similar productivities are obtained starting from the industrial by-product crude glycerol, demonstrating the possibilities of this hybrid heterogenenous biocatalyst for valorising bio-based raw materials.

En este proyecto hemos desarrollado una nueva generación de generadores bioinorgánicos para producir electricidad a partir de combustibles renovables. Estos sistemas se presentan como alternativa a las pilas de combustible basadas en procesos electroquímicos. En este contexto, hemos fabricado un nuevo sistema híbrido que fusiona una maquinaria biológica, capaz de transformar un combustible en energía mecánica (presión y vibración), con un material piezoeléctrico que transforma esa energía mecánica en electricidad (generador bioinorgánico).Los estímulos mecánicos de presión y vibración son generados gracias a la producción de gases a partir de combustibles. Esta reacción está catalizada por la maquinaria biológica (enzimas) que forma parte del generador. Estos dispositivos son capaces de producir electricidad a escala microscópica a partir de combustibles procedentes de fuentes renovables como azúcares y alcoholes provenientes de la biomasa. Este hecho es muy significativo, puesto que indirectamente permite generar electricidad con cero emisiones. Además, estos generadores bioinorgánicos abren un abanico apasionante de nuevas aplicaciones en campos tan diversos como la sensórica, la robótica y la energía.

En este proyecto hemos desarrollado una nueva generación de generadores bioinorgánicos para producir electricidad a partir de combustibles renovables. Estos sistemas se presentan como alternativa a las pilas de combustible basadas en procesos electroquímicos. En este contexto, hemos fabricado un nuevo sistema híbrido que fusiona una maquinaria biológica, capaz de transformar un combustible en energía mecánica (presión y vibración), con un material piezoeléctrico que transforma esa energía mecánica en electricidad (generador bioinorgánico).Los estímulos mecánicos de presión y vibración son generados gracias a la producción de gases a partir de combustibles. Esta reacción está catalizada por la maquinaria biológica (enzimas) que forma parte del generador. Estos dispositivos son capaces de producir electricidad a escala microscópica a partir de combustibles procedentes de fuentes renovables como azúcares y alcoholes provenientes de la biomasa. Este hecho es muy significativo, puesto que indirectamente permite generar electricidad con cero emisiones. Además, estos generadores bioinorgánicos abren un abanico apasionante de nuevas aplicaciones en campos tan diversos como la sensórica, la robótica y la energía.

En este proyecto hemos desarrollado una nueva generación de generadores bioinorgánicos para producir electricidad a partir de combustibles renovables. Estos sistemas se presentan como alternativa a las pilas de combustible basadas en procesos electroquímicos. En este contexto, hemos fabricado un nuevo sistema híbrido que fusiona una maquinaria biológica, capaz de transformar un combustible en energía mecánica (presión y vibración), con un material piezoeléctrico que transforma esa energía mecánica en electricidad (generador bioinorgánico).Los estímulos mecánicos de presión y vibración son generados gracias a la producción de gases a partir de combustibles. Esta reacción está catalizada por la maquinaria biológica (enzimas) que forma parte del generador. Estos dispositivos son capaces de producir electricidad a escala microscópica a partir de combustibles procedentes de fuentes renovables como azúcares y alcoholes provenientes de la biomasa. Este hecho es muy significativo, puesto que indirectamente permite generar electricidad con cero emisiones. Además, estos generadores bioinorgánicos abren un abanico apasionante de nuevas aplicaciones en campos tan diversos como la sensórica, la robótica y la energía.

Enzymes acting over glyceryl ethers are scarce in living cells, and consequently biocatalytic transformations of these molecules are rare despite their interest for industrial chemistry. In this work, we have engineered and immobilised a glycerol dehydrogenase from Bacillus stearothermophilus (BsGlyDH) to accept a battery of alkyl/aryl glyceryl monoethers and catalyse their enantioselective oxidation to yield the corresponding 3-alkoxy/aryloxy-1-hydroxyacetones. QM/MM computational studies decipher the key role of D123 in the oxidation catalytic mechanism, and reveal that this enzyme is highly enantioselective towards S-isomers (ee > 99%). Through structure-guided site-selective mutagenesis, we find that the mutation L252A sculpts the active site to accommodate a productive configuration of 3-monoalkyl glycerols. This mutation enhances the kcat 163-fold towards 3-ethoxypropan-1,2-diol, resulting in a specific activity similar to the one found for the wild-type towards glycerol. Furthermore, we immobilised the L252A variant to intensify the process, demonstrating the reusability and increasing the operational stability of the resulting heterogeneous biocatalyst. Finally, we manage to integrate this immobilised enzyme into a one-pot chemoenzymatic process to convert glycidol and ethanol into 3-ethoxy-1-hydroxyacetone and (R)-3-ethoxypropan-1,2-diol, without affecting the oxidation activity. These results thus expand the uses of engineered glycerol dehydrogenases in applied biocatalysis for the kinetic resolution of glycerol ethers and the manufacturing of substituted hydroxyacetones.

Feruloyl esterases synthesize butyl hydroxycinnamates, molecules possessing interesting biological properties, nonetheless, they exhibit a low stability under synthesis conditions in organic solvents, restricting its use. To enhance its operational stability in synthesis, we immobilized type A feruloyl esterase from Aspergillus niger (AnFAEA) using several carrier-bound and carrier-free strategies. The most active biocatalysts were: 1) AnFAEA immobilized on epoxy-activated carriers (protein load of 0.6 mgenzyme x mg−1carrier) that recovered 91 % of the initial hydrolytic activity, and 2) AnFAEA aggregated and cross-linked in the presence of 5 mg of BSA and 15 mM of glutaraldehyde (AnFAEA-amino-CLEAs), which exhibited 385 % of its initial hydrolytic activity; both using 4-nitrophenyl butyrate as substrate. The AnFAEA-amino-CLEAs were 12.7 times more thermostable at 60 °C than the AnFAEA immobilized on epoxy-activated carrier, thus AnFAEA-amino-CLEAs were selected for further characterization. Interestingly, during methyl sinapate hydrolysis (pH 7.2 and 30 °C), AnFAEA-amino-CLEAs KM was 15 % higher, while during butyl sinapate synthesis the KM was reduced in 63 %, both compared with the soluble enzyme. The direct esterification of butyl sinapate at solvent free conditions using sinapic acid 50 mM, reached 95 % conversion after 24 h employing AnFAEA-amino-CLEAs, which could be used for 10 cycles without significant activity losses, demonstrating their outstanding operational stability.

The immobilization of fluorescent proteins is a key technology enabling to fabricate a new generation of photoactive materials with potential technological applications. Herein we have exploited superfolder green (sGFP) and red (RFP) fluorescent proteins expressed with different polypeptide tags. We fused these fluorescent proteins to His-tags to immobilize them on graphene 3D hydrogels, and Cys-tags to immobilize them on porous microparticles activated with either epoxy or disulfide groups and with Lys-tags to immobilize them on upconverting nanoparticles functionalized with carboxylic groups. Genetically programming sGFP and RFP with Cys-tag and His-tag, respectively, allowed tuning the protein spatial organization either across the porous structure of two microbeads with different functional groups (agarose-based materials activated with metal chelates and epoxy-methacrylate materials) or across the surface of a single microbead functionalized with both metal-chelates and disulfide groups. By using different polypeptide tags, we can control the attachment chemistry but also the localization of the fluorescent proteins across the material surfaces. The resulting photoactive material formed by His-RFP immobilized on graphene hydrogels has been tested as pH indicator to measure pH changes in the alkaline region, although the immobilized fluorescent protein exhibited a narrower dynamic range to measure pH than the soluble fluorescent protein. Likewise, the immobilization of Lys-sGFP on alginate-coated upconverting nanoparticles enabled the infrared excitation of the fluorescent protein to be used as a green light emitter. These novel photoactive biomaterials open new avenues for innovative technological developments towards the fabrication of biosensors and photonic devices.

The immobilization of fluorescent proteins is a key technology enabling to fabricate a new generation of photoactive materials with potential technological applications. Herein we have exploited superfolder green (sGFP) and red (RFP) fluorescent proteins expressed with different polypeptide tags. We fused these fluorescent proteins to His-tags to immobilize them on graphene 3D hydrogels, and Cys-tags to immobilize them on porous microparticles activated with either epoxy or disulfide groups and with Lys-tags to immobilize them on upconverting nanoparticles functionalized with carboxylic groups. Genetically programming sGFP and RFP with Cys-tag and His-tag, respectively, allowed tuning the protein spatial organization either across the porous structure of two microbeads with different functional groups (agarose-based materials activated with metal chelates and epoxy-methacrylate materials) or across the surface of a single microbead functionalized with both metal-chelates and disulfide groups. By using different polypeptide tags, we can control the attachment chemistry but also the localization of the fluorescent proteins across the material surfaces. The resulting photoactive material formed by His-RFP immobilized on graphene hydrogels has been tested as pH indicator to measure pH changes in the alkaline region, although the immobilized fluorescent protein exhibited a narrower dynamic range to measure pH than the soluble fluorescent protein. Likewise, the immobilization of Lys-sGFP on alginate-coated upconverting nanoparticles enabled the infrared excitation of the fluorescent protein to be used as a green light emitter. These novel photoactive biomaterials open new avenues for innovative technological developments towards the fabrication of biosensors and photonic devices.

The immobilization of fluorescent proteins is a key technology enabling to fabricate a new generation of photoactive materials with potential technological applications. Herein we have exploited superfolder green (sGFP) and red (RFP) fluorescent proteins expressed with different polypeptide tags. We fused these fluorescent proteins to His-tags to immobilize them on graphene 3D hydrogels, and Cys-tags to immobilize them on porous microparticles activated with either epoxy or disulfide groups and with Lys-tags to immobilize them on upconverting nanoparticles functionalized with carboxylic groups. Genetically programming sGFP and RFP with Cys-tag and His-tag, respectively, allowed tuning the protein spatial organization either across the porous structure of two microbeads with different functional groups (agarose-based materials activated with metal chelates and epoxy-methacrylate materials) or across the surface of a single microbead functionalized with both metal-chelates and disulfide groups. By using different polypeptide tags, we can control the attachment chemistry but also the localization of the fluorescent proteins across the material surfaces. The resulting photoactive material formed by His-RFP immobilized on graphene hydrogels has been tested as pH indicator to measure pH changes in the alkaline region, although the immobilized fluorescent protein exhibited a narrower dynamic range to measure pH than the soluble fluorescent protein. Likewise, the immobilization of Lys-sGFP on alginate-coated upconverting nanoparticles enabled the infrared excitation of the fluorescent protein to be used as a green light emitter. These novel photoactive biomaterials open new avenues for innovative technological developments towards the fabrication of biosensors and photonic devices.

The immobilization of fluorescent proteins is a key technology enabling to fabricate a new generation of photoactive materials with potential technological applications. Herein we have exploited superfolder green (sGFP) and red (RFP) fluorescent proteins expressed with different polypeptide tags. We fused these fluorescent proteins to His-tags to immobilize them on graphene 3D hydrogels, and Cys-tags to immobilize them on porous microparticles activated with either epoxy or disulfide groups and with Lys-tags to immobilize them on upconverting nanoparticles functionalized with carboxylic groups. Genetically programming sGFP and RFP with Cys-tag and His-tag, respectively, allowed tuning the protein spatial organization either across the porous structure of two microbeads with different functional groups (agarose-based materials activated with metal chelates and epoxy-methacrylate materials) or across the surface of a single microbead functionalized with both metal-chelates and disulfide groups. By using different polypeptide tags, we can control the attachment chemistry but also the localization of the fluorescent proteins across the material surfaces. The resulting photoactive material formed by His-RFP immobilized on graphene hydrogels has been tested as pH indicator to measure pH changes in the alkaline region, although the immobilized fluorescent protein exhibited a narrower dynamic range to measure pH than the soluble fluorescent protein. Likewise, the immobilization of Lys-sGFP on alginate-coated upconverting nanoparticles enabled the infrared excitation of the fluorescent protein to be used as a green light emitter. These novel photoactive biomaterials open new avenues for innovative technological developments towards the fabrication of biosensors and photonic devices.

Understanding the functionality of immobilized enzymes with spatiotemporal resolution and under operando conditions is an unmet need in applied biocatalysis, as well as priceless information to guide the optimization of heterogeneous biocatalysts for industrial purposes. Unfortunately, enzyme immobilization still relies on trial-and-error approximations that prevail over rational designs. Hence, a modern fabrication process to achieve efficient and robust heterogeneous biocatalysts demands comprehensive characterization techniques to track and understand the immobilization process at the protein–material interface. Recently, our group has developed a new generation of self-sufficient heterogeneous biocatalysts based on alcohol dehydrogenases co-immobilized with nicotinamide cofactors on agarose porous microbeads. Harnessing the autofluorescence of NAD+(P)H and using time-lapse fluorescence microscopy, enzyme activity toward the redox cofactors can be monitored inside the beads. To analyze these data, herein we present an image analytical tool to quantify the apparent Michaelis–Menten parameters of alcohol dehydrogenases co-immobilized with NAD(P)+/H at the single-particle level. Using this tool, we found a strong negative correlation between the apparent catalytic performance of the immobilized enzymes and the bead radius when using exogenous bulky substrates in reduction reactions. Therefore, applying image analytics routines to microscopy studies, we can directly unravel the functional heterogeneity of different heterogeneous biocatalyst samples tested under different reaction conditions.

The immobilization of fluorescent proteins is a key technology enabling to fabricate a new generation of photoactive materials with potential technological applications. Herein we have exploited superfolder green (sGFP) and red (RFP) fluorescent proteins expressed with different polypeptide tags. We fused these fluorescent proteins to His-tags to immobilize them on graphene 3D hydrogels, and Cys-tags to immobilize them on porous microparticles activated with either epoxy or disulfide groups and with Lys-tags to immobilize them on upconverting nanoparticles functionalized with carboxylic groups. Genetically programming sGFP and RFP with Cys-tag and His-tag, respectively, allowed tuning the protein spatial organization either across the porous structure of two microbeads with different functional groups (agarose-based materials activated with metal chelates and epoxy-methacrylate materials) or across the surface of a single microbead functionalized with both metal-chelates and disulfide groups. By using different polypeptide tags, we can control the attachment chemistry but also the localization of the fluorescent proteins across the material surfaces. The resulting photoactive material formed by His-RFP immobilized on graphene hydrogels has been tested as pH indicator to measure pH changes in the alkaline region, although the immobilized fluorescent protein exhibited a narrower dynamic range to measure pH than the soluble fluorescent protein. Likewise, the immobilization of Lys-sGFP on alginate-coated upconverting nanoparticles enabled the infrared excitation of the fluorescent protein to be used as a green light emitter. These novel photoactive biomaterials open new avenues for innovative technological developments towards the fabrication of biosensors and photonic devices.

There is a current need to fabricate new biobased functional materials. Bottom-up approaches to assemble simple molecular units have shown promise for biomaterial fabrication due to their tunability and versatility for the incorporation of functionalities. Herein, the fabrication of catalytic protein thin films by the entrapment of catalase into protein films composed of a scaffolding protein is demonstrated. Extensive structural and functional characterization of the films provide evidence of the structural integrity, order, stability, catalytic activity, and reusability of the biocatalytic materials. Finally, these functional biomaterials are coupled with piezoelectric disks to fabricate a second generation of bio-inorganic generators. These devices are capable of producing electricity from renewable fuels through catalase-driven gas production that mechanically stimulates the piezoelectric material.

Sustainable electricity generation is one of the major current challenges for our society. In this context, the evolution of nanomaterials and nanotechnologies has enabled the fabrication of microscopic devices to produce clean energy from a great variety of renewable sources. To expand the possibilities of energy generation, we have designed and fabricated bioinorganic generators capable to produce electricity by conversion of chemical energy from renewable fuel sources. Unlike traditional generators, the systems described herein produce mechanical energy through enzyme-driven gas production which generates vibration and pressure that are thus converted into electricity by the action of a piezoelectric component properly integrated into the device. Our generators are able to produce an electric ernergy from different renewable sources like glucose, ethanol, and amino acids, attaining energy outputs around 250 nJ cm–2 and reaching maximum open-circuit voltages of up to 1 V. In addition, the produced energy can be easily regulated by adjusting both enzyme and fuel concentration which can tune the electrical output according to the application. The systems described herein propose a new concept for self-sufficient energy harvesting that bridges biocatalysis and piezoelectricity, where the energy production is based on the piezoelectric effect triggered by enzymatic action rather than on the enzyme-driven electron transfer that governs biofuel cells. Although the electric output is too low yet to be considered an alternative for energy production, this technology opens the door to power small devices. We envision the utilization of this technology in such remote locations where mechanical energy is lacking but there are chemical energy reservoirs.

Se presentan los primeros resultados de análisis isotópicos llevados a cabo a una selección de muestras de galena, goterones de plomo y objetos de base cobre del asentamiento fenicio de Abdera (Adra, Almería). Según los datos de isótopos de plomo, se pueden distinguir dos zonas diferentes de aprovisionamiento de mineral, Sierra de Gádor y la Cuenca de Vera (posiblemente Cerro Minado o Herrerías), todas ellas zonas mineras de la actual provincia de Almería (España). Los resultados ponen de relieve aspectos inéditos de gran interés sobre el comercio de metales entre los fenicios occidentales.

El presente documento tiene como base la problemática identificada por la administración municipal de Salento centrada en la deficiencia en los procesos organizativos y administrativos de las asociaciones que se dedican a la prestación del servicio de cabalgata en el casco urbano del municipio de Salento. Para caracterizar las variables vinculadas a dicha problemática se hizo una revisión de información secundaria sobre los antecedentes de la actividad en los archivos disponibles en la Secretaría de Turismo, deporte y cultura y en fuentes en línea de la alcaldía de Salento, la Corporación Autónoma Regional del Quindío - CRQ1 y la gobernación del Quindío. Se recopilo información primaria mediante la participación en diferentes reuniones realizadas en el centro administrativo municipal con los operadores de dicha actividad en los meses de diciembre del año 2016, enero y febrero de 2017, en las reuniones se confirmó el interés de la administración en acoger los resultados de este trabajo y se confirmó la disposición y apoyo de los operadores como actores claves en la construcción de alternativas de solución.

El presente documento tiene como base la problemática identificada por la administración municipal de Salento centrada en la deficiencia en los procesos organizativos y administrativos de las asociaciones que se dedican a la prestación del servicio de cabalgata en el casco urbano del municipio de Salento. Para caracterizar las variables vinculadas a dicha problemática se hizo una revisión de información secundaria sobre los antecedentes de la actividad en los archivos disponibles en la Secretaría de Turismo, deporte y cultura y en fuentes en línea de la alcaldía de Salento, la Corporación Autónoma Regional del Quindío - CRQ1 y la gobernación del Quindío. Se recopilo información primaria mediante la participación en diferentes reuniones realizadas en el centro administrativo municipal con los operadores de dicha actividad en los meses de diciembre del año 2016, enero y febrero de 2017, en las reuniones se confirmó el interés de la administración en acoger los resultados de este trabajo y se confirmó la disposición y apoyo de los operadores como actores claves en la construcción de alternativas de solución.

El presente documento tiene como base la problemática identificada por la administración municipal de Salento centrada en la deficiencia en los procesos organizativos y administrativos de las asociaciones que se dedican a la prestación del servicio de cabalgata en el casco urbano del municipio de Salento. Para caracterizar las variables vinculadas a dicha problemática se hizo una revisión de información secundaria sobre los antecedentes de la actividad en los archivos disponibles en la Secretaría de Turismo, deporte y cultura y en fuentes en línea de la alcaldía de Salento, la Corporación Autónoma Regional del Quindío - CRQ1 y la gobernación del Quindío. Se recopilo información primaria mediante la participación en diferentes reuniones realizadas en el centro administrativo municipal con los operadores de dicha actividad en los meses de diciembre del año 2016, enero y febrero de 2017, en las reuniones se confirmó el interés de la administración en acoger los resultados de este trabajo y se confirmó la disposición y apoyo de los operadores como actores claves en la construcción de alternativas de solución.

Enzyme immobilization can promote several advantages for their industrial application. In this work, a lipase from Hypocrea pseudokoningii was efficiently linked to four chemical supports: agarose activated with cyanogen bromide (CNBr), glyoxyl-agarose (GX), MANAE-agarose activated with glutaraldehyde (GA) and GA-crosslinked with glutaraldehyde. Results showed a more stable lipase with both the GA-crosslinked and GA derivatives, compared to the control (CNBr), at 50 °C, 60 °C and 70 °C. Moreover, all derivatives were stabilized when incubated with organic solvents at 50%, such as ethanol, methanol, n-propanol and cyclohexane. Furthermore, lipase was highly activated (4-fold) in the presence of cyclohexane. GA-crosslinked and GA derivatives were more stable than the CNBr one in the presence of organic solvents. All derivatives were able to hydrolyze sardine, açaí (Euterpe oleracea), cotton seed and grape seed oils. However, during the hydrolysis of sardine oil, GX derivative showed to be 2.3-fold more selectivity (eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) ratio) than the control. Additionally, the types of immobilization interfered with the lipase enantiomeric preference. Unlike the control, the other three derivatives preferably hydrolyzed the R-isomer of 2-hydroxy-4-phenylbutanoic acid ethyl ester and the S-isomer of 1-phenylethanol acetate racemic mixtures. On the other hand, GX and CNBr derivatives preferably hydrolyzed the S-isomer of butyryl-2-phenylacetic acid racemic mixture while the GA and GA-crosslink derivatives preferably hydrolyzed the R-isomer. However, all derivatives, including the control, preferably hydrolyzed the methyl mandelate S-isomer. Moreover, the derivatives could be used for eight consecutive cycles retaining more than 50% of their residual activity. This work shows the importance of immobilization as a tool to increase the lipase stability to temperature and organic solvents, thus enabling the possibility of their application

Optimal immobilization of enzymes on porous microbeads enables the fabrication of highly active and stable heterogeneous biocatalysts to implement biocatalysis in synthetic and analytical chemistry. However, empirical procedures for enzyme immobilization still prevail over rational ones because there is an unmet need for more comprehensive characterization techniques that aid to understand and trace the immobilization process. Here, we present the use of atomic force spectroscopy (AFS) as an innovative solution to indirectly characterize immobilized proteins on porous materials and monitor the immobilization process in real time. We investigate the mechanical properties of porous agarose microbeads immobilizing proteins by indenting a colloidal probe (silica microparticle) into a single bead. AFS demonstrates that the binding of proteins to the solid matrix of an agarose microbead alters its stiffness. Interestingly, we discovered that irreversible and multivalent immobilizations that make microbeads stiffer also stabilize the immobilized proteins against the temperature. Hence, we propose atomic force spectroscopy as a useful technique to indirectly unravel the stability of the immobilized enzymes investigating the mechanics of the heterogenous biocatalysts as a solid biomaterial beyond the intrinsic mechanics of the proteins.

Carrier-free immobilization of Candida rugosa lipase (CRL) and polymers containing primary amino groups were cross-linked using carbodiimide. To accomplish this, the free carboxyl groups of the enzyme were activated with carbodiimide-succinimide in organic medium, and then the activated proteins were cross-linked with different polyethylenimines (PEIs). The effect of the cross-linker chain length, the amount of added bovine serum albumin (BSA), and carbodiimide concentration on the catalytic properties of resulting cross-linked enzyme aggregates (CLEAs) was investigated. The CLEAs’ size, shape, specific activity, activity recovery, thermostability and enantioselectivity significantly varied according to the preparation procedure. The highest thermostable CRL-CLEA preparation was obtained with 1.3 kDa polyethyleneimine as cross-linker, 10 mg of BSA and 28 mM of carbodiimide. This preparation is 1.3-fold more active and thermostable than CLEAs prepared by the traditional method of amino cross-linking with glutaraldehyde, and retains 60% of residual activity after 22 h at 50 °C. Additionally, the CRL-CLEA preparation showed an enantioselectivity of 91% enantiomeric excess (ee). This immobilization procedure provides an alternative strategy for CLEA production, particularly for enzymes where the traditional method of cross-linking via lysine residues leads to enzyme inactivation.

The study of Phoenician metallurgy in the Iberian Peninsula context can be considered as a original research field in the whole Mediterranean, and specially in what concerns to the Phoenician culture. The lack of studies of metallurgical technologies related to Phoenician colonialism is evident, particularly in iron and plumb industries. Thus, in this essay we are presenting a study on Phoenitian metallurgy in the ancient city of Abdera, located in Cerro de Montecristo (Adra, Almería); therefore, we could set out a research field which could try to systematise the information related to metallurgy from the Phoenitian archaeological sites in the Iberian Peninsula. The methodology that we are using attempts to show new models of historical and archaeological research in the context of Empirical Sciences. The aim of this study is to obtain new information for understanding ancient metallurgy from a compositional-internal perspective—through specific methods like X Ray Diffraction., La metalurgia fenicia desarrollada en el contexto de la Península Ibérica puede considerarse un campo de investigación bastante novedoso en todo el ámbito mediterráneo y muy especialmente a lo que al pueblo fenicio se refiere. La falta de estudios exhaustivos sobre la tecnología metalúrgica introducida a través de la expansión colonial fenicia nos muestra una laguna en la historia de la tecnología, concretamente en prácticas industriales tan importantes como la siderurgia o la metalurgia del plomo. Por tanto, presentamos un estudio acerca de la metalurgia fenicia en la antigua ciudad de Abdera, ubicada en el Cerro de Montecristo (Adra, Almería), para, partiendo de un caso concreto, proponer en el futuro una línea de investigación que intente sistematizar la información relacionada con la metalurgia que hasta hoy han proporcionado los yacimientos fenicios peninsulares. La metodología empleada pretende mostrar modelos de indagación histórica y arqueológica en el marco de las Ciencias Empíricas que busca obtener nueva información que nos permita contemplar la metalurgia antigua desde una perspectiva interna – composicional – a través de métodos específicos como la Difracción de Rayos X.

The study of Phoenician metallurgy in the Iberian Peninsula context can be considered as a original research field in the whole Mediterranean, and specially in what concerns to the Phoenician culture. The lack of studies of metallurgical technologies related to Phoenician colonialism is evident, particularly in iron and plumb industries. Thus, in this essay we are presenting a study on Phoenitian metallurgy in the ancient city of Abdera, located in Cerro de Montecristo (Adra, Almería); therefore, we could set out a research field which could try to systematise the information related to metallurgy from the Phoenitian archaeological sites in the Iberian Peninsula. The methodology that we are using attempts to show new models of historical and archaeological research in the context of Empirical Sciences. The aim of this study is to obtain new information for understanding ancient metallurgy from a compositional-internal perspective—through specific methods like X Ray Diffraction., La metalurgia fenicia desarrollada en el contexto de la Península Ibérica puede considerarse un campo de investigación bastante novedoso en todo el ámbito mediterráneo y muy especialmente a lo que al pueblo fenicio se refiere. La falta de estudios exhaustivos sobre la tecnología metalúrgica introducida a través de la expansión colonial fenicia nos muestra una laguna en la historia de la tecnología, concretamente en prácticas industriales tan importantes como la siderurgia o la metalurgia del plomo. Por tanto, presentamos un estudio acerca de la metalurgia fenicia en la antigua ciudad de Abdera, ubicada en el Cerro de Montecristo (Adra, Almería), para, partiendo de un caso concreto, proponer en el futuro una línea de investigación que intente sistematizar la información relacionada con la metalurgia que hasta hoy han proporcionado los yacimientos fenicios peninsulares. La metodología empleada pretende mostrar modelos de indagación histórica y arqueológica en el marco de las Ciencias Empíricas que busca obtener nueva información que nos permita contemplar la metalurgia antigua desde una perspectiva interna – composicional – a través de métodos específicos como la Difracción de Rayos X.