Design of robust immobilized Xys1[delta] xylanase biocatalysts: Process intensification for XOS production from lignocellulosic residues

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 23-03-2018Esta tesis tiene embargado el acceso al texto completo hasta el 23-09-2019Xylo-oligosaccharides (XOS) have been described as prebiotics and display beneficial effects upon human health. The xylanase Xys1 , from Streptomyces halstedii JM8, was immobilized on glyoxyl-agarose beads by multipoint covalent attachment. The resulting immobilized Xys1 biocatalyst was 62-fold more thermo-stable than its soluble counterpart, and was used to catalyse the hydrolysis reaction of xylan for XOS production. A strategy based on surface coating with layers of polymers was developed to further increase the thermo-stability of this biocatalyst. The optimal modification consisted of surface coating with a bilayer formed by a layer of derived dextran polymer and a layer of polyethylenimine. The optimised biocatalyst was 550-fold more stable than the soluble Xys1 enzyme (at 70 ºC, pH 7). This optimised biocatalyst was used for production of xylo-oligosaccharides from soluble xylans of various sources in batch mode. Total reaction yields for beechwood, wheat and corncob xylan were 93% in 4 h, 44% in 5 h and 100% in 1 h, respectively. At these time points, xylan conversion yields to XOS were 65 % for beechwood xylan; 31% for wheat arabinoxylan; and 76% for corncob xylan. The optimised biocatalyst was reused for 15 reaction cycles of beechwood xylan hydrolysis without affecting its catalytic activity. To further increase the thermo-stability of the immobilized Xys1 biocatalyst, a strategy based on intensification of rigidification of the enzyme surface region involved in multipoint covalent attachment was developed. For this purpose, 1 4 additional lysine residues were introduced by substitution of native arginine residues to form 1 4 additional covalent bonds between enzyme and immobilization support. These covalent bonds, if formed, did not promote increased thermo-stabilizing effects, as they did not probably provide more intense rigidification of this enzyme surface area. Continuous flow reaction of corncob xylan hydrolysis for XOS production has been developed with an optimised packed-bed reactor (PBR). For this purpose, the xylanase Xys1 was immobilized by multipoint covalent immobilization on supports consisting of methacrylic polymer matrices previously functionalized with glyoxyl groups. The aim was to avoid enzyme leaching and to allow high flow rates since these supports display enhanced mechanical properties. The optimal support presented an area BET value of 69.5 m2/g and a narrow poresize distribution (80 nm pore size diameter). The resulting immobilized Xys1 biocatalyst onto this support displayed a maximum protein loading of 20 mg/g support, and high thermostability. A PBR was developed with this biocatalyst and used for continuous xylan hydrolysis at different flow rates. The specific volumetric productivity for XOS, 3,277 gXOS genzyme -1 h-1, was achieved at 10 mL/min flow rate with minimum production of xylose

Combined chemoenzymatic strategy for sustainable continuous synthesis of the natural product hordenine

To improve sustainability, safety and cost-efficiency of synthetic methodologies, biocatalysis can be a helpful ally. In this work, a novel chemoenzymatic stategy ensures the rapid synthesis of hordenine, a valuable phenolic phytochemical under mild working conditions. In a two-step cascade, the immobilized tyrosine decarboxylase from Lactobacillus brevis (LbTDC) is here coupled with the chemical reductive amination of tyramine. Starting from the abundant and cost-effective amino acid L-tyrosine, the complete conversion to hordenine is achieved in less than 5 minutes residence time in a fully-automated continuous flow system. Compared to the metal-catalyzed N,N-dimethylation of tyramine, this biocatalytic approach reduces the process environmental impact and improves its STY to 2.68 g/(L·h)

High Stabilization of Enzymes Immobilized on Rigid Hydrophobic Glyoxyl-Supports: Generation of Hydrophilic Environments on Support Surfaces

© 2020 by the authors.Very rigid supports are useful for enzyme immobilization to design continuous flow reactors and/or to work in non-conventional media. Among them, epoxy-methacrylic supports are easily functionalized with glyoxyl groups, which makes them ideal candidates for enzyme stabilization via multipoint covalent immobilization. However, these supports present highly hydrophobic surfaces, which might promote very undesirable effects on enzyme activity and/or stability. The hydrophilization of the support surface after multipoint enzyme immobilization is proposed here as an alternative to reduce these undesirable effects. The remaining aldehyde groups on the support are modified with aminated hydrophilic small molecules (glycine, lysine or aspartic acid) in the presence of 2-picoline borane. The penicillin G acylase from Escherichia coli (PGA) and alcohol dehydrogenase from Thermus thermophilus HB27 (ADH2) were immobilized on glyoxyl-functionalized agarose, Relizyme and Relisorb. Despite the similar density of aldehyde groups displayed by functionalized supports, their stabilization effects on immobilized enzymes were quite different: up to 300-fold lower by hydrophobic supports than by highly hydrophilic glyoxyl-agarose. A dramatic increase in the protein stabilities was shown when a hydrophilization treatment of the hydrophobic support surface was done. The PGA immobilized on the glyoxyl-Relisorb hydrophilized with aspartic acid becomes 280-fold more stable than without any treatment, and it is even more stable than the PGA immobilized on the glyoxyl agarose.This research was funded by the Spanish Ministry of Economy, Industry and Competitiveness (projects BIO2012-36861 and CTQ2015-70348) and the EU FP7 project SuSy (Sucrose Synthase as Cost-Effective Mediator of Glycosylation Reactions, C-KBBE/3293). Javier Rocha-Martin is grateful for the Juan de la Cierva fellowship (IJCI-2014-19260) funded by the Spanish Ministry of Economy, Industry and Competitiveness.Peer reviewe

Visualizing hydrophobic and hydrophilic enzyme interactions during immobilization by means of infrared microscopy

A novel Fourier transform infrared (FT-IR) microscopy method was developed and used to analyze the diffusion of lipase CalB in two different resins during immobilization. The method consisted of a streamlined sample preparation process and an automated transmission FT-IR microscopic measurement using a commercial benchtop device. The immobilization of CalB was performed on a hydrophobic resin containing aromatic groups (ECR1030M based on divinylbenzene) and on a hydrophilic resin containing ester groups and thus oxygen (ECR8204M based on methacrylate) and FT-IR revealed that the kinetic of immobilization and the distribution of the enzyme on the two resins were completely different. Furthermore, the technique revealed that CalB was immobilized on the external surface only in the case of the hydrophobic ECR1030M in a layer of about 50–70 μm, whereas when immobilized on the hydrophilic carrier ECR8204M the interaction of the enzyme with the carrier was uniform over the full diameter of the polymer bead. The enzyme activity however was higher on the hydrophobic support ECR1030M

Iron oxide nanoparticles: a platform for biomolecule conjugation

Among the nanomaterials, of great interest are iron oxide nanoparticles (IONPs), which possess unique magnetic properties, low toxicity and high biocompatibility. For these reason, IONPs are widely used in industrial and biomedical field. Here, IONPs were synthetized via co-precipitation method, obtaining NPs reproducible in shape and size distribution. First, IONPs were used as a platform for the conjugation of two thermophilic enzymes, L-aspartate oxidase and amylase to obtain an effective biocatalyst. Different binding strategies were studied leading to different enzymatic activity due to the different orientations and stretching of the proteins. Next, the NP-enzyme systems were nanoactuated by an alternate magnetic field (AMF) without increasing the overall temperature of the solution. Remarkably, the nano-systems were successfully reused for at three consecutive cycles of AMF with the loss of the 40% of the initial activity. IONPs were also used developed a nano-antibiotic system, using teicoplanin. The antimicrobial efficacy of NP-TEICO was assessed through classical microbiological methods and morphological studies. Results indicate that teicoplanin conjugation confers high and prolonged antimicrobial activity to IONPs toward Gram-positive bacteria, inhibiting also S. aureus biofilm formation, while no antimicrobial activity was detectable towards Gramnegative. Additionally, conjugation of teicoplanin improved the cytocompatibility of IONPs towards two human cell lines. To conclude, IONPs were successfully synthetized, functionalized and employed as a platform for biomolecules conjugation. Indeed two effective different nano-biocatalysts and a nano-antibiotic were obtained

Iron oxide nanoparticles: a platform for biomolecule conjugation

Among the nanomaterials, of great interest are iron oxide nanoparticles (IONPs), which possess unique magnetic properties, low toxicity and high biocompatibility. For these reason, IONPs are widely used in industrial and biomedical field. Here, IONPs were synthetized via co-precipitation method, obtaining NPs reproducible in shape and size distribution. First, IONPs were used as a platform for the conjugation of two thermophilic enzymes, L-aspartate oxidase and amylase to obtain an effective biocatalyst. Different binding strategies were studied leading to different enzymatic activity due to the different orientations and stretching of the proteins. Next, the NP-enzyme systems were nanoactuated by an alternate magnetic field (AMF) without increasing the overall temperature of the solution. Remarkably, the nano-systems were successfully reused for at three consecutive cycles of AMF with the loss of the 40% of the initial activity. IONPs were also used developed a nano-antibiotic system, using teicoplanin. The antimicrobial efficacy of NP-TEICO was assessed through classical microbiological methods and morphological studies. Results indicate that teicoplanin conjugation confers high and prolonged antimicrobial activity to IONPs toward Gram-positive bacteria, inhibiting also S. aureus biofilm formation, while no antimicrobial activity was detectable towards Gramnegative. Additionally, conjugation of teicoplanin improved the cytocompatibility of IONPs towards two human cell lines. To conclude, IONPs were successfully synthetized, functionalized and employed as a platform for biomolecules conjugation. Indeed two effective different nano-biocatalysts and a nano-antibiotic were obtained

Mejora de las propiedades de Redes Metal-Orgánicas mediante la asociación de nano-especies activas

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Químicas, Departamento de Química Inorgánica, leída el 22-11-2021Metal-Organic Frameworks (MOFs) are crystalline solids composed by inorganic units (atoms, clusters, chains, …) linked by ionocovalent bonds to organic polydentate ligands (carboxylates, phosphonates, azolates, …) procuring a highly porous three-dimensional (3D) network. From the first reports in the early 90s, interest in MOFs, their chemistry and their applications have increased exponentially. Proving so, are the >13,300 publications registered only in 2020 (Web-of-Science: “Metal-Organic Frameworks”). MOFs outstand from other porous materials due to their hybrid versatile organic-inorganic composition (tunable ligands, presence of unsaturated metallic centers…), their structural richness (multiple topologies, isoreticular families…) and their exceptional porosity (with specific surface areas-SBET up to 8000 m2·g-1). As consequence, they have become an ideal candidate for relevant industrial and societal applications...Las Redes Metal-Orgánicas o MOFs (por sus siglas en inglés: Metal-Organic Frameworks) son sólidos cristalinos formados a partir de unidades inorgánicas (átomos, clúster, cadenas, …) y ligandos orgánicos polidentados (carboxilatos, fosfonatos, azolatos, ...) creando una red tridimensional (3D) con una importante porosidad. Desde las primeras publicaciones en los años 90, ha crecido exponencialmente el interés en los MOFs, su química y sus aplicaciones. Muestra de ello son las más de 13300 publicaciones registradas tan sólo en 2020 (Web-of-Science: “Metal-Organic Frameworks”). Los MOFs destacan frente a otros materiales porosos debido a su composición híbrida versátil (ligando fácilmente modificable, presencia de centros metálicos insaturados), su riqueza estructural (multitud de topologías, familias isoreticulares) y su porosidad excepcional (áreas superficiales-SBET de hasta 8000 m2·g-1), convirtiéndolos en candidatos prometedores en diversas aplicaciones de relevancia social e industrial...Fac. de Ciencias QuímicasTRUEunpu