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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.

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.

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.