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Chitosan/silica hybrid powders, obtained by the sol-gel method (pH 6, 7, 8) are studied for proposing surface chemical models and rationalizing their behaviour as adsorbents. The chitosan and silica exposed groups in dried powders and their protonation state are inferred. Solid-state 29Si-NMR, XPS, zeta potential experiments and DFT computational calculations reveal the synthesis pH history of each sample. These details, summarized in surface models, are validated by copper adsorption: The pH 8 sample exhibits the highest hydrophilic and negative character, due to the deprotonated free silanol and neutral amine groups. These favor higher copper adsorption efficiency (30.4%) and loading capacity (0.077 mg Cu(II)/m2), through SiO−-Cu(II) electrostatic interactions and [Cu-(NH2)x]2+ complexation. For lower synthesis-pH samples, the neutral silanol and protonated amine populations increase, and the net surface charge becomes positive. This reduces the copper adsorption through electrostatic repulsions. The protonated amine and deprotonated silanol groups are the main responsible of electrokinetic surface response of materials prepared at lower and higher pH, respectively. The complexation is also involved in copper interactions, especially in surfaces populated by neutral amines. In conclusion, this work helps to understand the pH-dependent surface activity of chitosan/hybrid materials, being helpful for their further functionalization and applications.

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 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-...

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.

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 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.

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 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.

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 characterize the micelles and particles in the reaction mixtures during the synthesis of SBA-3 type silica at different temperatures, acid and salt concentrations. The adsorption of the siliceous species on the micelles leads to their transformation into wormlike micelles which aggregate into “liquid particles” (gyroids or spheres, observed by optical microscopy) through the screening of the micellar charges. Their transformation into “solid particles” occurs through the replacement of the intermicellar hydrogen and Van der Waals bonds involving the silanol groups by siloxane bonds.

Spherical particles (∅>3 μm, with 5–10 nm pore size) of SBA15-type mesoporous silica, usable as stationary phase for HPLC, were prepared with tetraethoxysilane (TEOS) as silica source, triblock copolymer Pluronic P123 (EO20PO70EO20) as surfactant S0 and cetyltrimethylammonium bromide (CTMABr) as co-surfactant S+. The synthesis mechanism involves a surfactant-silica species self-assembly process in acidic medium under quiescent conditions, where S0H3O+X−I+ and S+X−I+ interactions occur. The syntheses were carried out using two procedures: (a) with one heating step and (b) with two heating steps. Synthesis conditions, such as the dilution, temperature and acidity of the reaction mixture and the temperature and duration of the reaction, influence the porous characteristics as well as the morphology and size of the particles. Spherical particles were obtained with one heating step under conditions that weaken the surfactants-silicate interactions such as high dilution, high temperature and low acidity. The modification of the micelle volume with the temperature and acidity allowed the adjustment of the pore size.

Chitosan was derivatized by two methodologies for analyzing their effect on chitosan physicochemical characteristics and its applicability as carrier for Bacillus circulans b-galactosidase immobilization. Glutaraldehyde (GA) and epichlorohydrin (EPI) were used for crosslinking and activation of chitosan, producing the corresponding supports (C-GA and C-EPI-EPI) after a one-step and a two- step process, respectively. The spherical shape and mean diameter of chitosan particles was not significantly affected by polymer derivati- zation, while Fourier transform infrared analysis showed that in both cases, chitosan polymer was chemically modified. TGA analysis indicated that C-EPI-EPI was the most thermally stable. The high degree of activation of C-EPI-EPI (586 lmol of aldehydes/g) resulted in the highest loss of activity during immobilization; hence a support with 100 lmol of aldehydes/g was produced (C-EPI-EPI100). The highest expressed activity (89.3 IU/g) was obtained with the enzyme immobilized in C-GA, while the biocatalyst with highest thermal stability at 60 � C was obtained with C-EPI-EPI100 (half-life was 84-fold higher than the one of the soluble enzyme). The best compro- mise between biocatalyst expressed activity and thermal stability corresponded to b-galactosidase immobilized in C-EPI-EPI100. Accord- ing to this study, chitosan derivatized with EPI is a thermally stable carrier appropriate for producing highly stable immobilized B. circulans b-galactosidase. V C 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 40171.

β-galactosidase from Kluyveromyces lactis was covalently immobilised on a Glyoxyl Sepharose (GS) support by multi-point attachment. The enzyme immobilisation process was very efficient; the supports immobilised almost all the protein responsible for the catalytic activity in a short period of time, retaining approximately 82% of the activity in the case of the optimal immobilised preparations. Stability of the GS derivatives varied as a function of enzyme-support incubation time. The optimal immobilised preparation was produced after 2 h of incubation with the support at alkaline pH. This derivative, obtained by multi-point covalent attachment, was 100-fold more stable at pH 7 and 50 °C than the cyanogen bromide Sepharose derivative obtained by a one-point covalent immobilisation method. Stabilisation was also observed under a wide range of experimental conditions. This method allowed the immobilisation of 9000 IU enzyme g−1 of support, resulting in highly active and stable derivatives suitable for industrial processes., We thank COLCIENCIAS, Municipio de Rionegro and Universidad de Antioquia for financial support through the program “Becas doctorales 2007 en el convenio de cooperación # 030-2006”. We also thank the Spanish Ministry of Science for financial support through intramural projects (2008801058) and project No. (AGL-2009- 07625).

International audience; The formation mechanism of large defect cavities (∅ 11-23 nm) beside the MCM41 mesopores (∅ ∼ 2.3 nm), was studied from mixtures of sodium silicate, cetyl trimethyl ammonium bromide (CTAB) and ethyl acetate (EtAc) with the molar composition 1 SiO2; 1 Na2O; x CTAB; y EtAc: 237 H2O (with x = 0.33 or 0.65, y = 3, 6, 9 or 15). The silica flocculation and polycondensation were obtained by decreasing the pH using the hydrolysis of different amounts of EtAc. The reaction was carried out in two steps at 25 and 80 °C. The samples were characterized by SEM, TEM, XRD, nitrogen adsorption and TGA. The CTA/SiO2 molar ratio, which forms complete silica layers covering the micelles in the MCM41 structure, was estimated from the area covered by the CTA cations in the micelles head and the silica tetrahedron area in the layers. The number of silicate anions needed to compensate the CTA cations charge was determined in function of the pH from the anions ionization constants. From these two parameters, a model, showing the percentage of missing tetrahedra in the silica layers, was established in function of the pH and the CTA/SiO2 molar ratio, and used to propose a mechanism which explains the formation of large defect cavities. Their size was discussed taking into account the pH decrease rate, which is function of the EtAc amount. The particles morphology, varying from long and curved prismatic rods to isometric particles of strongly bended short rods, is due to their softness, which results of the presence of the numerous defect cavities.

Monomodal or bimodal porous silicas with large mesopores, constituted by particles or having a monolithic (block type) morphology, respectively, are synthesized using sodium silicate as siliceous species source, cetyltrimethylammonium bromide (CTAB) as pore template and ethyl acetate (EtAc) as pH modifier. The monomodal porosity is represented by 20-30 nm pores and the bimodal one by these pores and also macropores. These characteristics are modulated in function of the CTAB and EtAc concentrations as well as the pH and hydrothermal treatment. The role of these reagents upon the porosity is rationalized. The presence of high CTAB concentration and a rather low pH decreasing rate (function of EtAc concentration and hydrothermal treatment) are essential for having the already known bimodal mesoporous silicas (BMS). On the contrary a rather high pH decreasing rate promotes the formation of the new bimodal mesoporous-macroporous silicas (BMMS) synthesized in this work, where the EtAc also plays the role of emulsion forming agent. The hydrolytic stability of the synthesized silica under aqueous conditions, at different pH values, makes these silicas good candidates for application in different areas of catalysis, especially in the enzymatic one.

The use of hard templates is one of the best ways for obtaining ordered mesoporous carbons (OMC) with well controlled morphology and porosity, characteristics that are very important for their use in many technological applications. Since the SBA-16 type mesoporous silica has a 3D connected mesostructure and can be synthesized with different morphologies, we select this material as a hard template and study the influence of its pore (cavities and entrances) size and hydrothermal stability on the qualities of the OMC replicas obtained by liquid impregnation of sucrose.

The evolution of the species in acidic solutions, prepared from tetraethoxysilane (TEOS), surfactant (Cetyltrimethylammonium bromide, Pluronic P123 or Pluronic F127) and HCl, was followed by visual observation and Dynamic Light Scattering (DLS) measurements between 25 and 90°C until their precipitation. The nature of the precipitated phases was studied by optical microscopy (in-situ in the reaction mixtures) and by SEM (after filtration and drying). In all cases, the first stage corresponds to the formation of micelles coated by siliceous species (composite colloids). In a second stage the modification of their colloidal characteristics leads to their aggregation with a phase separation into micron-sized liquid particles. In the third stage, the progress of the polycondensation transforms the latter into solid particles of mesoporous silica. The final morphology depends on the polycondensing degree of the silica before the phase separation and on the prevailing synthesis parameters (temperature and pH).

Procedures reported in the literature to modify the silanol content of mesoporous silica are applied to a material prepared with the triblock copolymer Pluronic F127 in acidic medium under quiescent conditions, in order to adapt it for silylation reaction. These include: calcination at relatively high temperature and rehydroxylation in presence of NH4OH. The nature and content of silanol groups in these materials is measured by following the pyridine interaction with them through the infrared specific absorptions at 1595 and 1446 cm−1 assigned to pyridine interacting with hydrogen-bonded and free silanol groups respectively. In order to render these SBA-16-type mesoporous silica hydrophobic, a silylation reaction is effected in which its efficiency depends mainly on the nature and concentration of silanol groups. To follow the degree of silylation a method based on infrared transmission spectroscopy (FTIR) is designed in this work. With this method, unlike solid state 29Si-NMR, it is possible to measure the relative concentrations of the organic groups bonded to silica when two silylating reagents are used, and to qualify in an indirect way how the weak silanol groups change during the different post synthesis treatments of a mesoporous silica.

The relations between the morphology (particle shape and size) of SBA-15 and SBA-16 mesoporous silica and the synthesis parameters (composition of the reaction mixtures, nature of the triblock surfactant, fluoride anions, synthesis procedure) were established. This allowed defining the conditions for the formation of materials with micro-sized isometric particles suitable for HPLC applications. The presence of fluoride anions favors the formation of spherical shaped particles, with probably higher weak acidic silanol groups content to give higher hydrophobic materials after silylation, which is an advantage for their use as HPLC reverse phases. The different materials were evaluated in HPLC tests (separation of mixtures of aromatic and polynuclear aromatic molecules).

Mesoporous silica is used as filler for styrene-butadiene rubber (SBR); filler-polymer interactions are compared with those exhibited when Ultrasil silica (VN3) is used. A silane coupling agent is added to improve filler dispersion and its influence on the bound-rubber formation is also investigated. The bound-rubber content is higher for the mesoporous silica and increases further for the sample containing silane. The increase is explained by chemical interactions between filler and rubber and penetration of the rubber chains into the mesopores. This is confirmed by 13C solid-state NMR, IR spectroscopy and differential scanning calorimetry. Dynamic mechanical thermal analysis shows higher storage modulus for the rubber filled with mesoporous silica.