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The life of RNA polymerase II (RNAPII) transcripts is shaped by the dynamic formation of mutually exclusive ribonucleoprotein complexes (RNPs) that direct transcript biogenesis and turnover. A key regulator of RNA metabolism in the nucleus is the scaffold protein ARS2 (arsenic resistance protein 2), bound to the cap binding complex (CBC). We report here that alternative splicing of ARS2′s intron 5, generates cytoplasmic isoforms that lack 270 amino acids from the N-terminal of the protein and are functionally distinct from nuclear ARS2. Switching of ARS2 isoforms within the CBC in the cytoplasm has dramatic functional consequences, changing ARS2 from a NMD inhibitor to a NMD promoter that enhances the binding of UPF1 to NCBP1 and ERF1, favouring SURF complex formation, SMG7 recruitment and transcript degradation. ARS2 isoform exchange is also relevant during arsenic stress, where cytoplasmic ARS2 promotes a global response to arsenic in a CBC-independent manner. We propose that ARS2 isoform switching promotes the proper recruitment of RNP complexes during NMD and the cellular response to arsenic stress. The existence of non-redundant ARS2 isoforms is relevant for cell homeostasis, and stress response.

The diffusional behaviour of H2 adsorbed in a novel mesoporous N-doped carbon material and on exfoliated graphite has been studied with quasi-elastic neutron scattering. While the activation energies for diffusion obtained for exfoliated graphite was in line with prior results, H2 in the mesoporous carbon showed an exceptionally low diffusion barrier and behaved qualitatively like liquid hydrogen. Complementary results from neutron and X-ray diffraction as well as from isothermal nitrogen sorption studies provide a full characterisation of this novel mesoporous carbon.

Inefficient autologous tissue recovery in diverse skin injuries increases the susceptibility of patients to infections caused by multiresistant microorganisms, resulting in a high mortality rate. Nonviral transfection is an attractive alternative for these patients, where genetically modified cells incorporated into skin substitutes could release additional antimicrobial agents into the native skin. In this work, we have modulated the conditions of using a nonviral system for transfection of primary human keratinocytes and fibroblasts, consisting of a polymer/plasmid DNA (pDNA) complex called polyplex and its effects on the expression of LL-37 antimicrobial peptide. Linear and branched polyethylenimine (PEI) polymers in different weight concentrations were varied for evaluating the formation and colloidal characteristics of the polyplexes. The PEI/pDNA polyplexes with 19 nitrogen/phosphate ratio are nanometric particles (400 and 250 nm with linear and branched PEI, respectively) exhibiting positive surface (+30 ± 2 mV). Both kinds of polyplexes allowed the expression of a reporter gene and increased the human cathelicidin antimicrobial peptide gene expression in transfected keratinocytes and fibroblasts; however, greater cytotoxicity was observed when polyplexes formed with branched PEI were used. Moreover, cell culture supernatants from transfected cells with linear PEI/pDNA polyplexes showed enhanced antimicrobial activity (decrease of bacterial growth in 95.8%) against a

During a developmental period that extends postnatally in the mouse, proliferating multipotent retinal progenitor cells produce one of 7 major cell types (rod, cone, bipolar, horizontal, amacrine, ganglion, and Müller glial cells) as they exit the cell cycle in consecutive waves. Cell production in the retina is tightly regulated by intrinsic, extrinsic, spatial, and temporal cues, and is coupled to the timing of cell cycle exit. Arsenic-resistance protein 2 (ARS2, also known as SRRT) is a component of the nuclear cap-binding complex involved in RNA Polymerase II transcription, and is required for cell cycle progression. We show that postnatal retinal progenitor cells (RPCs) require ARS2 for proper progression through S phase, and ARS2 disruption leads to early exit from the cell cycle. Furthermore, we observe an increase in the proportion of cells expressing a rod photoreceptor marker, and a loss of Müller glia marker expression, indicating a role for ARS2 in regulating cell fate specification or differentiation. Knockdown of Flice Associated Huge protein (FLASH), which interacts with ARS2 and is required for cell cycle progression and 3′-end processing of replication-dependent histone transcripts, phenocopies ARS2 knockdown. These data implicate ARS2–FLASH-mediated histone mRNA processing in regulating RPC cell cycle kinetics and neuroglial cell fate specification during postnatal retinal development.

Chitosan-silica materials offer a specific environment for the adsorption of biofunctional molecules, such as the antimicrobial peptide KR-12. The objective here is to rationalize the changes in the physicochemical properties of these chitosan-silica materials in function of the synthesis pH, using 0.02 w/v % chitosan as catalyst and aggregation agent and, to correlate these characteristics with the loading/delivery and activity/stability of KR-12 antimicrobial peptide. The CS-6 material, prepared at pH 6, exhibits higher surface area (745 m2/g) and total pore volume (0.58 cm3/g) due to the lower incorporation of chitosan swollen chains (9.36 wt %). Higher pHs produced denser materials (CS-7 and CS-8) with higher entangled chitosan incorporated (> 17 wt%). Adsorption of KR-12 peptide was found to take two different mechanisms depending on the chitosan-silica support, Langmuir-type for CS-6 material and Freundlich-type for CS-7 and CS-8 materials. According to the KR-12 release profiles, more hydrophobic interactions were observed in the CS-6 material exposing Lys and Arg residues from the peptide towards the material surface. This was correlated with the higher antimicrobial activity of this material against S. aureus strain (MIC = 128 μg/mL). Additionally, the KR-12 peptide adsorbed in the CS-6 hybrid support is 34 % more protected from the proteolytic action of α-chymotrypsin than the free one. In conclusion, the proposed models of different porous environments in the studied chitosan-silica materials supports the KR-12 peptide loading/delivery mechanisms, leading to biofunctionalized solid antimicrobial materials exhibiting proteolytic stability. This knowledge is useful for designing new antibacterial materials for biomedical applications. Keywords: Chitosan-silica particle mechanism formation, KR-12 peptide adsorption/delivery mechanisms, KR-12 controlled release, KR-12/chitosan-silica antimicrobial solid materials, Proteolysis stability

The research of simple and fast enzyme immobilization methods, preserving the enzyme activity and improving the thermal stability, is in the spotlight. The objective of this work is to develop a β-galactosidase immobilization one-pot route, combining the silica sol-gel encapsulation (SSGE) process with a metal chelation strategy by using chitosan and Ca

Multifunctional and biocompatible hydrogels are on the focus of wound healing treatments. Protein and polysaccharides silica hybrids are interesting wound dressing alternatives. The objective of this review is to answer questions such as why silica for wound dressings reinforcement? What are the roles and contributions of silane precursors and silica on the functional properties of hydrogel wound dressings? The effects of tailoring the porous, morphological, and chemical characteristics of synthetic silicas on the bioactivity of hybrid wound dressings hydrogels are explored in the first part of the review. This is followed by a commented review of the mechanisms of silica/protein and silica/polysaccharide interactions and their impact on the barrier, scaffold, and delivery matrix functions of the derived hydrogels. Such information has important consequences for wound healing and paves the way to multidisciplinary researches on the production, processing, and biomedical application of this kind of hybrid materials.

The spontaneous aggregation of chitosan and carboxymethylchitosan polymers can be advantageous for the enzyme confinement on these colloidal systems during immobilization processes. The initial crucial step involves the polymer-enzyme adduct formation. The objective here is to determine the interactions that drive the adduct formation between these polymers and β-galactosidase from Bacillus circulans. The chemical characterization of chitosan and its carboxymethyl-derivate allowed to explain their colloidal behavior and design the four-unit fragments ligands used for the docking study. The deacetylation degree (0.6 times lower), isoelectric point (5.2 instead 6.4) and substitution degree (DSO = 1.779 and DS2N = 0.441) of carboxymenthylchitosan are due to the hydroxide concentration (>25%) and 30 °C modification conditions. Favorable Van der Waals and H-bond interactions between chitosan-β-galactosidase and contribution of electrostatic attraction mediated by calcium ions for carboxymethylchitosan-β-galactosidase explained the zeta potential and dynamic light scattering results at pH 7.0. These interactions occur onto the external surface of this galactosidase, without affecting the catalytic activity. A cross-linked enzyme aggregates-type model was proposed for the formation of the adducts, based on the complementary experimental-docking results. They contribute understanding the behavior of polyelectrolyte chitosan-derived matrices for enzyme immobilization.

The research of simple and fast enzyme immobilization methods, preserving the enzyme activity and improving the thermal stability, is in the spotlight. The objective of this work is to develop a β-...

Simple Summary Currently, radiotherapy and chemotherapy are the most commonly used options, in addition to surgery, to treat cancer. There has been tremendous progress in interfacing nanotechnology to current cancer therapeutic protocols. For example, nanoparticles are used as drug carriers in chemotherapy and as radiation dose enhancers in radiotherapy. However, most of the work to date has been focused on tumor cells. To make significant progress in this field, we need to consider the tumor microenvironment, especially cancer-associated fibroblast cells that promote tumor growth. Our study shows the potential of targeting both tumor cells and cancer-associated fibroblasts to reap the full benefits of cancer nanomedicine. Abstract Due to recent advances in nanotechnology, the application of nanoparticles (NPs) in cancer therapy has become a leading area in cancer research. Despite the importance of cancer-associated fibroblasts (CAFs) in creating an optimal niche for tumor cells to grow extensively, most of the work has been focused on tumor cells. Therefore, to effectively use NPs for therapeutic purposes, it is important to elucidate the extent of NP uptake and retention in tumor cells and CAFs. Three tumor cell lines and three CAF cell lines were studied using gold NPs (GNPs) as a model NP system. We found a seven-fold increase in NP uptake in CAFs compared to tumor cells. The retention percentage of NPs was three-fold higher in tumor cells as compared to CAFs. Furthermore, NP uptake and retention were significantly enhanced using a 50 nM concentration of docetaxel (DTX). NP uptake was improved by a factor of three in tumor cells and a factor of two in CAFs, while the retention of NPs was two-fold higher in tumor cells compared to CAFs, 72 h post-treatment with DTX. However, the quantity of NPs in CAFs was still three-fold higher compared to tumor cells. Our quantitative data were supported by qualitative imaging data. We believe that targeting of NPs in the presence of DTX is a very promising approach to accumulate a higher percentage of NPs and maintain a longer retention in both tumor cells and CAFs for achieving the full therapeutic potential of cancer nanotechnology.

Fibrin is a protein-based hydrogel formed during blood coagulation. It can also be produced in vitro from human blood plasma, and it is capable of resisting high deformations. However, after each deformation process, it loses high amounts of water, which subsequently makes it mechanically unstable and, finally, difficult to manipulate. The objective of this work was to overcome the in vitro fibrin mechanical instability. The strategy consists of adding silica or chitosan-silica materials and comparing how the different materials electrokinetic-surface properties affect the achieved improvement. The siliceous materials electrostatic and steric stabilization mechanisms, together with plasma protein adsorption on their surfaces, were corroborated by DLS and ζ-potential measurements before fibrin gelling. These properties avoid phase separation, favoring homogeneous incorporation of the solid into the forming fibrin network. Young’s modulus of modified fibrin hydrogels was evaluated by AFM to quantitatively measure stiffness. It increased 2.5 times with the addition of 4 mg/mL silica. A similar improvement was achieved with only 0.7 mg/mL chitosan-silica, which highlighted the contribution of hydrophilic chitosan chains to fibrinogen crosslinking. Moreover, these chains avoided the fibroblast growth inhibition onto modified fibrin hydrogels 3D culture observed with silica. In conclusion, 0.7 mg/mL chitosan-silica improved the mechanical stability of fibrin hydrogels with low risks of cytotoxicity. This easy-to-manipulate modified fibrin hydrogel makes it suitable as a wound dressing biomaterial.

En la síntesis de 4-metoxicinamoilglicerol, se aprovecha el subproducto de biodiesel para obtener un filtro UV hidrofílico, derivado de cinamato, útil en formulaciones de bloqueadores solares. El objetivo de este trabajo fue demostrar que la esterificación del ácido 4-metoxicinámico y el glicerol, mediado por la lipasa inmovilizada de Thermomyces lanuginosus, es selectiva hacia el monoester del filtro UV 4-metoxicinamoilglicerol, cuyas características químicas favorecen la formación de nanopartículas, por gelificación ionotrópica en N-succinil-quitosano. Una conversión de ácido cinámico ~34% en hexano es mayor que los valores ya reportados, sin la presencia de otros subproductos o productos de degradación. Esto facilita, el proceso de purificación por extracción líquido-líquido. Las entidades de glicerilo libre favorecen su incorporación en nanopartículas de N-succinil-quitosano, con un tamaño de alrededor de 185±77nm, que son promisorias para los productos de protección solar. The synthesis of 4-methoxycinnamoylglycerol takes advantage of the biodiesel subproduct for obtaining a hydrophilic UV cinnamate derivate fi lter, useful in sunscreen formulations. The objective here was to demonstrate that esterifi cation of 4-methoxycinnamic acid and glycerol mediated by immobilized-lipase from Thermomyces lanuginosus is selective towards 4-methoxycinnamoylglycerol monoester UV fi lter, whose chemical characteristics favor the nanoparticles formation by ionotropic gelation on N-Succinyl chitosan. A cinnamic acid conversion ∼ 34% in hexane is higher than in other reports, without the presence of other sub-products or degradation products. This eases the purifi cation process by liquid esterification reaction, because it is thermophilic and active in a wide pH range (Fernandez-Lafuente, 2010). Its immobilization has improved its catalytic behavior in this kind of reaction due to the interfacial activation (Bassi et al. 2016; Zhou et al. 2012), thermal and conformational stabilization (Bernal et al. 2018; Matte et al. 2014) conferred by the support. Moreover, its immobilization by one-pot silica sol-gel process in the presence of polyethilenimine (PEI) is so simple for obtaining a biocatalyst with high protein load (90%), encapsulation yield (47%) and 247-times more stable at 65°C than the soluble enzyme (Escobar et al. 2018), which could increase the conversion degree of 4-MCA and glycerol during the esterification reaction at 65ºC. This immobilized-lipase has been not tested yet for production of 4-MCG

Biophysical characterization of antimicrobial peptides helps to understand the mechanistic aspects of their action. The physical behavior of the KR-12 antimicrobial peptide (e.g. orientation and changes in secondary structure), was analyzed after interactions with a Staphylococcus aureus membrane model and solid surfaces. We performed antimicrobial tests using Gram-positive S. aureus (ATCC 25923) bacteria. Moreover, Langmuir-Blodgett experiments showed that the synthetic peptide can disturb the lipidic membrane at a concentration lower than the Minimum Inhibitory Concentration, thus confirming that KR-12/lipid interactions are involved. Partially- and fully-deactivated KR-12 hybrid samples were obtained by physisorption and covalent immobilization in chitosan/silica and glyoxal-rich solid supports. The correlation of Langmuir-Blodgett data with the α-helix formation, followed by FTIR-ATR in a frozen-like state, and the antimicrobial activity showed the importance of these interactions and conformation changes on the first step action mode of this peptide. This is the first time that material science (immobilization in solid surfaces assisted by FTIR-ATR analysis in frozen-like state) and physical (Langmuir-Blodgett/Schaefer) approaches are combined for exploring mechanistic aspects of the primary action mode of the KR-12 antimicrobial peptide against S. aureus.

Silica-based sol-gel enzyme immobilization aided by polyamines has been used for generating active and stable heterogeneous biocatalysts. However, the encapsulation mechanism and its influence on the characteristics of the resulting biocatalysts have not been fully understood yet. This work aimed to shed light on how Thermomyces lanuginosus lipase, siliceous species and the additive polyethylenimine (PEI) interact during enzyme encapsulation, by establishing a correlation between addition of PEI, material formation and enzyme encapsulation. The addition of PEI appears to increase enzyme incorporation producing a biocatalyst with high encapsulation efficiency in terms of percentage of encapsulated protein (90%) and activity (47%). Furthermore, we observed that the addition of PEI resulted in a biocatalyst with high catalytic activity (600 U/g) and thermal stability (247-fold more stable than the soluble enzyme at 65 °C). The formation of silica particles was followed over time, showing a homogeneous incorporation of the enzyme in its active form. Intrinsic fluorescence spectroscopy suggested that the addition of PEI during the encapsulation process resulted in a conservation of the structural and functional features of the enzyme, suggesting a stabilizing role of the additive. The integration of the results of this work allowed proposing a polycondensation/enzyme encapsulation mechanism for silica-based sol-gel encapsulation mediated by PEI. These new evidences will contribute to the design of novel and more efficient silica-based biocatalysts achieved by sol gel encapsulation methodology.

The objective of this paper was to carry out an integral study of the use of hydrophobic chitosan as a low-cost support for immobilizing lipases and their further application in the selective hydrolysis of fish oil. Chitosan functionalized with different alkyl chains (C4, C8, C12) were characterized by FTIR, TGA, SEM, and Rose Bengal adsorption. Lipase B from Candida antarctica (CalB) and lipase from Rhizomucor miehei (RML) were immobilized obtaining a higher expressed activity at a longer alkyl chain length of support. Biocatalyst thermal stability showed that the impact of the alkyl chain length on enzyme stabilization varied according to the lipase source. The biocatalysts were applied in menhaden oil hydrolysis. Total polyunsaturated fatty acids released after 30 h of reaction with lipases immobilized in butyl, octyl and dodecyl-chitosan was 60, 107, and 90 mM for CalB biocatalysts, and 560, 392, and 50 mM for RML biocatalysts, respectively. Selectivity of CalB was not affected by the alkyl chain, while in the case of RML, a higher selectivity to cis-4,7,10,13,16,19-docohexaenoic acid release was obtained with dodecyl-chitosan. In conclusion, the adequate functionalization of chitosan varied according to lipase source, affecting their activity, stability and performance in the hydrolysis of fish oil.

A sustainable industrial application of the enzymes requires their reuse and stability. These objectives are accomplished here for the Thermomyces lanuginosus lipase and Bacillus subtilis protease, by immobilization in porous silica modified with (3-aminopropyl) triethoxysilane. This silylation leaves aminopropyl groups on the silica surface, leading to a heterofunctional support, with amine and alkyl entities that participate in ionic exchange-hydrophobic interactions, respectively, with the enzyme. In this work, the silica surface is effectively modified, as it is shown by FTIR and 29 Si- and 13 C-ssNMR spectroscopic techniques. The characteristics of the support surface explain the changes of the catalytic properties of the two immobilized enzymes. This support promotes the homogeneous orientation and stabilization of the enzyme during the one-pot immobilization process, without the costs of other heterofunctiontal materials obtained by multiple functionalization steps. Both immobilized enzymes show thermal stabilization in aqueous solution (25 mM sodium phosphate buffer, pH 7.0) at 60 °C and in 100% acetone at 40 °C, without addition of stabilizers. Both biocatalysts exhibit high performance on the synthesis of sugar fatty acid esters, in comparison with others, reported under similar reaction conditions. Due to the characteristic of the lipase/aminopropyl silica and protease/aminopropyl silica biocatalysts, they are promisors in applications that need high mechanical and enzymatic stability.

The interactions between Thermomyces lanuginosus lipase (TLL) and phenyl silica-based supports affect the immobilization mechanisms and their catalytic behavior. The modulation of phenyl groups density on the silica surface and porous characteristics were determined by TGA, FTIR, 29Si NMR and N2 adsorption porosimetry. The correlation of the affinity constant and maximum adsorption capacity with the lixiviation results allowed determine differences in the enzyme adsorption mechanism in function of the immobilization pH and phenyl groups density. In the support with low phenyl groups density, the adsorption of a higher amount of enzyme is promoted. However, the pore confinement and the microenvironment generate decrease expressed activity. This can be due to the stiffness and structural changes of the adsorbed enzymes, which were studied by following the thermal stability at 65°C, protein distribution, kinetic parameters and diffusion restrictions. The biocatalyst prepared on support with low density of phenyl groups at pH6.0, exhibits the best balance between expressed activity, thermal stability and immobilization efficiency. This due to homogeneous distribution of the enzyme in the support with phenyl groups, which increases the affinity of the enzyme by the substrate, even the diffusion restrictions decrease the Vmax. These results contribute to rationalize the effects of the immobilization conditions and supports type on its catalytic behavior.

A new strategy for functionalization of porous silica with phenyl-methyl groups is developed and demonstrated in this work. Bare silica, a hierarchical bimodal porous material, was functionalized with non-hydrolysable alkylsilane (trimethylphenylsilane) by liquid phase grafting, without the use of additional catalyst. The analyses carried out by FTIR, zeta potential and 29Si NMR of modified material indicating that the silane is efficiently and stably adsorbed on silica surface. Computational simulations showing the interaction between silane and silica is by formation of bridges between phenyl ring of silane and silanol groups of silica, with interaction energies similar to those found for hydrogen bridges. Additionally, this modification generates a change on hydrophobicity of silica (11% more hydrophobic than bare silica) that allows the success immobilization of lipase from Thermomyces lanuginosus, used as probe biomolecule. This biocatalyst exhibits a specific activity of 45.7 IU (50% more than those obtained with bare silica) and a half-life of 89.9 h at 45 °C in acetone, which is 5-fold higher than for the biocatalyst prepared with the bare silica. These results could contribute to the preparation of new and efficient materials to be used as supports for biocatalysts, delivery drugs and remediation.

Improving the enzyme stability is a challenge for allowing their practical application. The surfactants are stabilizing agents, however, there are still questions about their influence on enzyme properties. The structure-activity/stability relationship for β-galactosidase from Bacillus circulans is studied here by Circular Dichroism and activity measurements, as a function of temperature and pH. The tendency of preserving the β-sheet and α-helix structures at temperatures below 65 °C and different pH is the result of the balance between the large- and short-range effects, respecting to the active site. This information is fundamental for explaining the structural changes of this enzyme in the presence of Triton X-100 surfactant and ethanol. The enzyme thermal stabilization in the presence of this surfactant responds to the rearrangement of the secondary structure for having optimal activity/stability. The effect of ethanol is more related with changes in the dielectric properties of the aqueous solution than with protein structural transformations. These results contribute to understand the effects of surfactant-enzyme interactions on the enzyme behavior, from the structural point of view and to rationalize the surfactant-based stabilizing strategies for β-galactosidades.

The biomineralisation of metal phosphates is a promising approach to develop more efficient nanobiocatalysts; however, the interactions between the protein and the inorganic mineral are poorly understood. Elucidating which protein regions most likely participate in the mineral formation will guide the fabrication of more efficient biocatalysts based on metal-phosphate nanoflowers. We have biomineralised the lipase from Thermomyces lanuginosus using three calcium, zinc and copper phosphates to fabricate different types of bio-inorganic nanoflowers. To better understand how the biomineralisation process affects the enzyme properties, we have computationally predicted the protein regions with a higher propensity for binding Ca2+, Cu2+ and Zn2+. These binding sites can be considered as presumable nucleation points where the biomineralisation process starts and explain why different metals can form bio-inorganic nanoflowers of the same enzyme with different functional properties. The formation of calcium, copper and zinc phosphates in the presence of this lipase gives rise to nanoflowers with different morphologies and different enzymatic properties such as activity, stability, hyperactivation and activity–pH profile; these functional differences are supported by structural studies based on fluorescence spectroscopy and can be explained by the different locations of the predicted nucleation sites for the different metals. Among the three metals used herein, the mineralisation of this lipase with zinc-phosphate enables the fabrication of bio-inorganic nanoflowers 34 times more stable than the soluble enzyme. These bio-inorganic nanoflowers were reused for 8 reaction cycles achieving 100% yield in the hydrolysis of p-nitrophenol butyrate but losing more than 50% of their initial activity after 6 operational cycles. Finally, this heterogeneous biocatalyst was more active and enantioselective than the soluble enzyme (ee = 79%(R)) towards the kinetic resolution of rac-1-phenylethyl acetate yielding the R enantiomer with ee = 84%.

Los organil-amino-alcoxi-silanos son moléculas interesantes para la síntesis de materiales híbridos. Su uso se ha visto limitado debido a la escasa oferta comercial y a su baja estabilidad. En este trabajo se prepararon tres fenil-amino-dietoxi-silanos mediante condensación de fenil-cloro-dietoxi-silano y aminas (ruta 1) y fenil-trietoxi-silano y amiduros de magnesio (ruta 2). La caracterización y los estudios de estabilidad de los compuestos fueron llevados a cabo por 1H-RMN y CG-MS. Ambas rutas presentaron rendimientos mayores a 91%. Fue posible establecer que la sal acida, obtenida en la ruta 1, cataliza la descomposición total de los productos en un tiempo inferior a dos semanas. El medio de reacción de la ruta 2 mostró ser mucho menos agresivo, y después de dos semanas, aún permanece más del 60% de la concentración inicial. Los organil-amino-alcoxi-silanos son moléculas interesantes para la síntesis de materiales híbridos. Su uso se ha visto limitado debido a la escasa oferta comercial y a su baja estabilidad. En este trabajo se prepararon tres fenil-amino-dietoxi-silanos mediante condensación de fenil-cloro-dietoxi-silano y aminas (ruta 1) y fenil-trietoxi-silano y amiduros de magnesio (ruta 2). La caracterización y los estudios de estabilidad de los compuestos fueron llevados a cabo por 1H-RMN y CG-MS. Ambas rutas presentaron rendimientos mayores a 91%. Fue posible establecer que la sal acida, obtenida en la ruta 1, cataliza la descomposición total de los productos en un tiempo inferior a dos semanas. El medio de reacción de la ruta 2 mostró ser mucho menos agresivo, y después de dos semanas, aún permanece más del 60% de la concentración inicial.

The enzyme encapsulation is a very well‐known stabilization pathway. However, there are some challenges in order to avoid the enzyme denaturation under encapsulation conditions. The β‐galactosidase from Bacillus circulans was immobilized through sol‐gel encapsulation route assisted by Triton X‐100 surfactant and sugars. The effects of sugar presence in the immobilization process and the gelation time on the biocatalyst activity/stability were explained taking into account the characteristics of the formed silica matrix and the changes of the enzyme environment. The enzyme was effectively immobilized by this strategy, with high immobilization yield in terms of activity (29%) and expressed activity (47 IU/g). The immobilization through silica sol‐gel in the presence of 1×10−3 M Triton X‐100 and fructose conferred 28.4‐fold higher stability to the enzyme compared with the soluble form. This is an advantage for its use in the synthesis of the galacto‐oligosaccharides at 50ºC. The total lactose conversion to galacto‐oligosaccharides was 26%wt, which is comparable with that reported in the literature. The obtained biocatalyst is useful for the synthesis of galacto‐oligosaccharides and its catalytic behavior is rationalized in this work.

Currently, the development of multifunctional materials for targeted/controlled delivery of multiple dietary supplements from a single dosage form calls strongly the attention. Chitosan, silica, and derived materials have been reported as matrices for β-galactosidase and, they also can be silicon and calcium sources. There are synthesis strategies for obtaining multifunctional products containing these compounds. They would avoid the usual consumption of diverse supplements by people suffering from digestive disorders, such as lactose intolerance and their concomitant effects on bone mineralization and hard tissue maintenance. The objective of this review is to analyze the chemical aspects involved in the synthesis, processing and gastrointestinal transit of this kinds of multifunctional product. It will help to answer critical research questions related to the drawbacks, challenges, developments, and opportunities that these materials offer as matrices for oral transport and controlled delivery of functional β-galactosidase and silicon/calcium minerals up to the intestine.

The lipase and Triton X-100 mixture is common for stabilization, immobilization and application processes of these kinds of enzymes. The objective of this article was to study the structural behavior and catalytic performance of Thermomyces lanuginose lipase in the presence of Triton X-100 at 25 °C and different pHs. The structural changes were followed by circular dichroism, correlating them with the catalytic performance, which is reported as the initial lipase activity in the hydrolysis of p‑nitro phenyl butyrate at zero time and residual activity after 48 h of incubation in the absence or presence of surfactant, at the selected pHs. Computational simulations allowed to explain the correlations between the physicochemical changes and the formation of surfactant protein complex, leading to the elucidation of the main interactions that drive activity and stability of this lipase in presence of the Triton X-100 surfactant. Main results showed the Triton X-100-enzyme complex modulates the site active geometry, favoring a better substrate-enzyme adjustment, which influences the activity and stability at evaluated pHs. This study contributes to understand the effect of some additives commonly used to improve the biocatalytic performance on several applications for different industrial fields.

The sol-gel process has been very useful for preparing active and stable biocatalysts, with the possibility of being reused. Especially those based on silica are well known. However, the study of the enzyme behavior during this process is not well understood until now and more, if the surfactant is involved in the synthesis mixture. This work is devoted to the encapsulation of β-galactosidase from Bacillus circulans in silica by sol-gel process, assisted by non-ionic Triton X-100 surfactant. The correlation between enzyme activity results for the β-galactosidase in three different environments (soluble in buffered aqueous reference solution, in the silica sol, and entrapment on the silica matrix) explains the enzyme behavior under stress conditions offered by the silica sol composition and gelation conditions. A stable β-galactosidase/silica biocatalyst is obtained using sodium silicate, which is a cheap source of silica, in the presence of non-ionic Triton X-100, which avoids the enzyme deactivation, even at 40 °C. The obtained biocatalyst is used in the whey hydrolysis for obtaining high value products from this waste. The preservation of the enzyme stability, which is one of the most important challenges on the enzyme immobilization through the silica sol-gel, is achieved in this study.

This chapter describes the production of biocatalysts based on silica and related inorganic materials. The emphasis is put on the use of hierarchical porous silica for encapsulation of large enzymes such as β-galactosidases. These as-synthesized materials are used for enzyme immobilization by adsorption. However, they are chemically modified for improving the enzyme thermal stability and avoiding lixiviation. These materials and encapsulating routes are expanded to other enzymes, like lipases. At the end of the chapter, the future of the composite carbon/silica materials is envisaged.

Two carbonaceous–siliceous composite materials, produced by hydrothermal and carbonization processes, were evaluated as immobilization support for lipase from Alcaligenes sp. These materials exhibited similar chemical characteristics but their carbon content and porous characteristics were different, which explain the catalytic behavior and stability of the biocatalysts immobilized on them. Higher activity and immobilization selectivity was achieved with the microporous material that had higher carbon content. The lipase immobilized on the mesoporous material had a higher thermal stability at 55 °C, pH 7.0 or at 40 °C in tert-butanol, simulating the reaction conditions required for organic synthesis. Both biocatalysts were tested in the synthesis of palmitoyl ascorbate and they were compared with the commercial biocatalyst QLC. The synthesis conversions with the lipase immobilized in mesoporous materials and with the biocatalyst QLC were similar (50%), but only the former could be reused. These are promising biocatalysts for industrial applications.

The use of aniline as carbon source has two advantages for the synthesis of carbon materials exhibiting conducting properties: the aromatic ring contributes to the graphitic character and the presence of nitrogen could work as n-dopant, decreasing the band gap of the carbon materials. These conditions contribute to improve their efficiency as electrocatalysts, for example in fuel cells. The objective of this work is to correlate physicochemical characteristics and electrical behavior of carbon samples prepared by nanocasting of aniline in mesoporous silica and its subsequent carbonization under controlled conditions.

The use of heterogeneous biocatalysis in industrial applications is advantageous and the enzyme stability improvement is a continuous challenge. Therefore, we designed β-galactosidase heterogeneous biocatalysts by immobilization, involving the support synthesis and enzyme selection (from Bacillus circulans, Kluyveromyces lactis, and Aspergillus oryzae). The underivatized, tailored, macro-mesoporous silica exhibited high surface area, offered high enzyme immobilization yields and activity. Its chemical activation with glyoxyl groups bound the enzyme covalently, which suppressed lixiviation and conferred higher pH and thermal stability (120-fold than for the soluble enzyme), without observable reduction of activity/stability due to the presence of silica. The best balance between the immobilization yield (68%), activity (48%), and stability was achieved for Bacillus circulans β-galactosidase immobilized on glyoxyl-activated silica, without using stabilizing agents or modifying the enzyme. The enzyme stabilization after immobilization in glyoxyl-activated silica was similar to that observed in macroporous agarose-glyoxyl support, with the reported microbiological and mechanical advantages of inorganic supports. The whey lactolysis at pH 6.0 and 25°C by using this catalyst (1 mg ml−1, 290 UI g−1) was still 90%, even after 10 cycles of 10 min, in batch process but it could be also implemented on continuous processes at industrial level with similar results.

The kinetic study of the colloidal and enzymatic stability for the β-galactosidase of Bacillus circulans was carried out in function of the presence of Triton X-100 surfactant, under orbital agitation and varying the pH and temperature. The correlation between the Dynamic Light Scattering and enzyme assay data, supported by z-potential and Differential Scanning Calorimetry analyses, gave insights about the mechanism of the protective role of the surfactant against the enzyme deactivation during its incubation. The best conditions for preserving the enzymatic activity, under orbital agitation, were: presence of 1 × 10−3M Triton X-100, at pH 6.0 and 25 °C or 40 °C during less than 24 h, even in the presence of 0.1 M sodium cations or 4% ethanol. As these conditions also affect the polycondensation of the siliceous species and the enzyme-silica interactions, these could be considered as primary information for designing and optimizing an encapsulation route of β-galactosidase in silica, by a sol–gel process assisted by surfactant.

Polyaniline-silica composites made with mesoporous silica have been reported to improve the mechanical properties of the free polymer. However, there are not many studies about the effect of the chemical and physical properties of the inorganic material used as hosts over the final properties of polyaniline and the composites. Here, polyaniline-silica composites, exhibiting electrical conductivity, were synthesized using different types of SBA-16 mesoporous silica as hosts. The effects of the characteristics of the hosts in the polymerization kinetic, molecular weight and electrical conductivity of the final polymer were studied. The composites showed higher electrical conductivity in comparison with polyaniline synthesized under similar conditions of this work but in the absence of silica. The use of silica allowed to obtain composites with defined morphology, which did not occur in the absence of these hosts. From the results of this work, it can be concluded that SBA-16 mesoporous silica hosts have a bifunctional role in the formation of polyaniline-silica composites: the hosts behaved like nucleation sites, which favors polymerization of high molecular weight polyaniline and they catalyzed the polymerization reaction, especially when aluminum is present in the structure of the host.

β-Galactosidase from Bacillus circulans was immobilized on hierarchical macro-mesoporous silica by multipoint covalent attachment by formation of Schiff bases between enzyme and support. The enzyme was effectively immobilized with high yields (around 60–80%) and expressed activity (around 50–80%) depending on the concentration of aldehyde groups in the carrier. Immobilization of β-galactosidase in chemically modified silica conferred excellent thermal stability to the biocatalyst and enzyme leaching was completely avoided. The effect of the concentration of functional groups in the silica surface was studied on the activity and thermal stability of the biocatalyst. The best hybrid catalyst was 370-fold more stable than the soluble enzyme at pH 6 and 55 °C.

Hierarchical porous silica, particles or monoliths, were synthesized by polycondensation of sodium silicate in the presence of cetyltrimethylammonium bromide and ethyl acetate at different concentrations under hydrothermal conditions. They were used as the support for the immobilization of β-galactosidase from Kluyveromyces lactis by adsorption. The enzyme loading capacity (higher than 50 mg g−1 support) and the retention ability (lixiviation less than 20 % after 72 h of catalysis) of these supports were explained as a function of the hierarchical porosity, mesopore sizes of 10–40 nm, and macropore sizes of 0.07–20 μm and the presence of ionized silanol groups on the surface. The optimum pH value and temperature for the maximum activity of the obtained hybrid biocatalyst were evaluated, indicating that the three-dimensional structure of the lactase was not significantly affected during the immobilization process. The stability under extreme conditions was improved in comparison with soluble lactase. The porous supports exhibited morphological and porous stability under the immobilization and catalytic processes. These results show that the obtained materials are good candidates for the immobilization of large enzymes, such as β-galactosidase.

Dynamic light scattering (DLS) was used to follow the evolution of the micelles of two triblock copolymer surfactants, Pluronic F127 and Pluronic P123, in diluted aqueous reaction mixtures during the formation of respectively SBA-16 and SBA-15 type mesoporous silica particles at different temperatures, acid and salt concentrations. The silica source (tetraethoxysilane TEOS) is adsorbed by the micelles and undergoes hydrolysis. The resulting siliceous species coat the hydrophilic corona of the micelles, transforming those into composite colloids. The shape of the colloids formed from Pluronic F127 remains spherical but their size increases, whereas the spherical shape of the colloids with Pluronic P123 evolutes into an elongated shape. The decrease of the repulsion energy of these composite colloids allows their aggregation into “liquid particles” (spheres or polyhedra), as observed by optical microscopy. The “liquid particles”, which can be re-dissolved in the case of the SBA-16 precursor by decreasing the temperature, transform into the final solid mesoporous silica particles by siloxane bonds formation within the coalesced silica walls.

The mesoporous silica have been considered fascinating materials for many techonological applications due to their porous and morphological characteristics. This review focuses on their use as stationary phases for liquid chromatography, supports for immobilizing biomolecules, catalysts, agent for polymer reinforcement and hard templates for the preparation of mesoporous carbons.