Response surface methodology as an approach to determine optimal activities of lipase entrapped in sol-gel matrix using different vegetable oils

The conditions for maximization of the enzymatic activity of lipase entrapped in sol-gel matrix were determined for different vegetable oils using an experimental design. The effects of pH, temperature, and biocatalyst loading on lipase activity were verified using a central composite experimental design leading to a set of 13 assays and the surface response analysis. For canola oil and entrapped lipase, statistical analyses showed significant effects for pH and temperature and also the interactions between pH and temperature and temperature and biocatalyst loading. For the olive oil and entrapped lipase, it was verified that the pH was the only variable statistically significant. This study demonstrated that response surface analysis is a methodology appropriate for the maximization of the percentage of hydrolysis, as a function of pH, temperature, and lipase loading.

Investigation of single tryptophan proteins encapsulated in TEOS-derived sol-gel matrices by fluorescence spectroscopy /

In the work reported here, optically clear, ultrathin TEOS derived sol-gel slides which were suitable for studies of tryptophan (Trp) fluorescence from entrapped proteins were prepared by the sol-gel technique and characterized. The monitoring of intrinsic protein fluorescence provided information about the structure and environment of the entrapped protein, and about the kinetics of the interaction between the entrapped protein and extemal reagents. Initial studies concentrated on the single Trp protein monellin which was entrapped into the sol-gel matrices. Two types of sol-gel slides, termed "wet aged", in which the gels were aged in buffer and "dry-aged", in which the gels were aged in air , were studied in order to compare the effect of the sol-gel matrix on the structure of the protein at different aging stages. Fluorescence results suggested that the mobility of solvent inside the slides was substantially reduced. The interaction of the entrapped protein with both neutral and charged species was examined and indicated response times on the order of minutes. In the case of the neutral species the kinetics were diffusion limited in solution, but were best described by a sum of first order rate constants when the reactions occurred in the glass matrix. For charged species, interactions between the analytes and the negatively charged glass matrix caused the reaction kinetics to become complex, with the overall reaction rate depending on both the type of aging and the charge on the analyte. The stability and conformational flexibility of the entrapped monellin were also studied. These studies indicated that the encapsulation of monellin into dry-aged monoliths caused the thermal unfolding transition to broaden and shift upward by 14°C, and causedthe long-term stability to improve by 12-fold (compared to solution). Chemical stability studies also showed a broader transition for the unfolding of the protein in dry-aged monoliths, and suggested that the protein was present in a distribution of environments. Results indicated that the entrapped proteins had a smaller range of conformational motions compared to proteins in solution, and that entrapped proteins were not able to unfold completely. The restriction of conformational motion, along with the increased structural order of the internal environment of the gels, likely resulted in the improvements in themial and long-term stability that were observed. A second protein which was also studied in this work is the metal binding protein rat oncomodulin. Initially, the unfolding behavior of this protein in aqueous solution was examined. Several single tryptophan mutants of the metal-binding protein rat oncomodulin (OM) were examined; F102W, Y57W, Y65W and the engineered protein CDOM33 which had all 12 residues of the CD loop replaced with a higher affinity binding loop. Both the thermal and the chemical stability were improved upon binding of metal ions with the order apo < Ca^^ < Tb^"^. During thermal denaturation, the transition midpoints (Tun) of Y65W appeared to be the lowest, followed by Y57W and F102W. The placement of the Trp residue in the F-helix in F102W apparently made the protein slightly more thermostable, although the fluorescence response was readily affected by chemical denaturants, which probably acted through the disruption of hydrogen bonds at the Cterminal end of the F-helix. Under both thermal and chemical denaturation, the engineered protein showed the highest stability. This indicated that increasing the number of metal ligating oxygens in the binding site, either by using a metal ion with a higher coordinatenumber (i.e. Tb^*) which binds more carboxylate ligands, or by providing more ligating groups, as in the CDOM33 replacement, produces notable improvements in protein stability. Y57W and CE)OM33 OM were chosen for further studies when encapsulated into sol-gel derived matrices. The kinetics of interaction of terbium with the entrapped proteins, the ability of the entrapped protein to binding terbium, as well as thermal stability of these two entrapped protein were compared with different levels of Ca^"*^ present in the matrix and in solution. Results suggested that for both of the proteins, the response time and the ability to bind terbium could be adjusted by adding excess calcium to the matrix before gelation. However, the less stable protein Y57W only retained at most 45% of its binding ability in solution while the more stable protein CDOM33 was able to retain 100% binding ability. Themially induced denaturation also suggested that CDOM33 showed similar stability to the protein in solution while Y57W was destabilized. All these results suggested that "hard" proteins (i.e. very stable) can easily survive the sol-gel encapsulation process, but "soft" proteins with lower thermodynamic stability may not be able to withstand the sol-gel process. However, it is possible to control many parameters in order to successfully entrap biological molecules into the sol-gel matrices with maxunum retention of activity.

Sol-gel entrapped biosystems: enzyme(s) and whole cells

In this work two different procedures to utilize the sol-gel technology were applied to immobilize/encapsulate enzymes and living cells. CO2 has reached levels in the atmosphere that make it a pollutant. New methods to utilize this gas to obtain products of added value can be very important, both from an environmentally point of view and from an economic standpoint. The first goal of this work was to study the first reaction of a sequential, three-step, enzymatic process that carries out the conversion of CO2 to methanol. Of the three oxidoreductases involved, our focus was on formate dehydrogenase (FateDH) that converts CO2 to formate. This reaction requires the presence of the cofactor β-nicotinamide adenine dinucleotide in reduced form (NADH). The cofactor is expensive and unstable. Our experiments were directed towards generating NADH from its oxidized form (NAD+), using glutamate dehydrogenase (GDH). The formation of NADH from NAD+ in aqueous medium was studied with both free and sol-gel entrapped GDH. This reaction was then followed by the conversion of CO2 to formate, catalysed by free or sol-gel entrapped FateDH. The quantification of NADH/NAD+ was made using UV/Vis spectroscopy. Our results showed that it was possible to couple the GDH-catalyzed generation of the cofactor NADH with the FateDH-catalyzed conversion of CO2, as confirmed by the detection of formate in the medium, using High Performance Liquid Chromatography (HPLC). The immobilization of living cells can be advantageous from the standpoint of ease of recovery, reutilization and physical separation from the medium. Also dead cells may not always exhibit enzymatic activities found with living cells. In this work cell encapsulation was performed using Escherichia coli bacteria. To reduce toxicity for living organisms, the sol-gel method was different than for enzymes, and involved the use of aqueous-based precursors. Initial encapsulation experiments and viability tests were carried out with E. coli K12. Our results showed that sol-gel entrapment of the cells was achieved, and that cell viability could be increased with additives, namely betaine that led to greater viability improvement and was selected for further studies. For an approach to “in-cell” Nuclear Magnetic Resonance (NMR) experiments, the expression of the protein ctCBM11 was performed in E. coli BL21. It was possible to obtain an NMR signal from the entrapped cells, a considerable proportion of which remained alive after the NMR experiments. However, it was not possible to obtain a distinctive NMR signal from the target protein to distinguish it from the other proteins in the cell.

Influence of the use of Aliquat 336 in the immobilization procedure in sol-gel of lipase from Bacillus sp ITP-001

Aliquat 336, a liquid hydrophobic material, was used at different concentrations (0.5-3.0%, w/v) as an additive in the preparation of encapsulated lipase from Bacillus sp. ITP-001 on sol-gel silica matrices using tetraethoxysilane (TEOS) as the precursor. The resulting hydrophobic matrices and immobilized lipases were characterized with regard to specific surface area (BET method), adsorption-desorption isotherms, pore volume (Vp) and size (dp) by nitrogen adsorption (BJH method) and scanning electron microscopy (SEM). The catalytic activities and the corresponding coupling yields were assayed in the hydrolysis of olive oil. In comparison with pure silica matrices, the immobilization process in the presence of Aliquat 336 decreased the values for specific surface area and increased the values for pore specific volume (Vp) and mean pore diameter (dp). This behavior may be related to the partial adsorption of the enzyme on the external surface of the hydrophobic matrix as indicated by scanning electron microscopy. Aliquat 336 concentrations in the range from 0.5 to 1.5% (w/v) provided immobilized derivatives with higher coupling yields and better substrate affinity. The highest coupling yield (Y-A = 71%) was obtained for the immobilized enzyme prepared in the presence of 1.5% Aliquat which gave the following morphological properties: specific surface area = 183 m(2)/g, pore specific volume (Vp) = 0.36 cc/g and mean pore diameter (dp)= 91 angstrom. (c) 2012 Elsevier B.V. All rights reserved.

Enhancement of the Electrochemical Performance of SWCNT dispersed in a Silica Sol-gel Matrix by Reactive Insertion of a Conducting Polymer

The electroassisted encapsulation of Single-Walled Carbon Nanotubes was performed into silica matrices (SWCNT@SiO2). This material was used as the host for the potentiostatic growth of polyaniline (PANI) to yield a hybrid nanocomposite electrode, which was then characterized by both electrochemical and imaging techniques. The electrochemical properties of the SWCNT@SiO2-PANI composite material were tested against inorganic (Fe3+/Fe2+) and organic (dopamine) redox probes. It was observed that the electron transfer constants for the electrochemical reactions increased significantly when a dispersion of either SWCNT or PANI was carried out inside of the SiO2 matrix. However, the best results were obtained when polyaniline was grown through the pores of the SWCNT@SiO2 material. The enhanced reversibility of the redox reactions was ascribed to the synergy between the two electrocatalytic components (SWCNTs and PANI) of the composite material.

Síntese e estudo de ortossilicatos de zinco luminescentes com aplicação da técnica sol-gel

Usually, the concepts of the Sol-Gel technique are not applied in experimental chemistry courses. This work presents a feasible experiment for chemistry instruction, which involves the synthesis of luminescent materials - Zn2SiO4, with and without Mn2+ as a dopant - by the Sol-Gel technique. The obtained materials were analyzed by scanning electron microscopy, X-Ray diffraction, IR spectroscopy and luminescence measures by UV-vis spectroscopy. The results allow the students to confirm the luminescent properties of the zinc orthosilicate luminophores as well as the structural features expected from literature data.

Synthesis and biocompatibility of an experimental glass ionomer cement prepared by a non-hydrolytic sol-gel method

The aims of this study were to demonstrate the synthesis of an experimental glass ionomer cement (GIC) by the non-hydrolytic sol-gel method and to evaluate its biocompatibility in comparison to a conventional glass ionomer cement (Vidrion R). Four polyethylene tubes containing the tested cements were implanted in the dorsal region of 15 rats, as follows: GI - experimental GIC and GII - conventional GIC. The external tube walls was considered the control group (CG). The rats were sacrificed 7, 21 and 42 days after implant placement for histopathological analysis. A four-point (I-IV) scoring system was used to graduate the inflammatory reaction. Regarding the experimental GIC sintherization, thermogravimetric and x-ray diffraction analysis demonstrated vitreous material formation at 110oC by the sol-gel method. For biocompatibility test, results showed a moderate chronic inflammatory reaction for GI (III), severe for GII (IV) and mild for CG (II) at 7 days. After 21 days, GI presented a mild reaction (II); GII, moderate (III) and CG, mild (II). At 42 days, GI showed a mild/absent inflammatory reaction (II to I), similar to GII (II to I). CG presented absence of chronic inflammatory reaction (I). It was concluded that the experimental GIC presented mild/absent tissue reaction after 42 days, being biocompatible when tested in the connective tissue of rats.

Morphological and mechanical properties of hybrid matrices of polysiloxane-polyvinyl alcohol prepared by sol-gel technique and their potential for immobilizing enzyme

Hybrid matrices of polysiloxane-polyvinyl alcohol (POS-PVA) were prepared by sol-gel technique using different concentrations of the organic component (polyvinyl alcohol, PVA) in the synthesis medium. The goal was to prepare carriers for immobilizing enzyme by taking into consideration properties as hardness, mean pore diameter, specific surface area and pore size distribution. The matrices were activated with sodium metaperiodate to render functional groups for binding the lipase from Candida rugosa, used here as a study model. Results showed that low proportion of PVA gave POS-PVA with low surface area and pore volume, although with higher hardness. The chemical activation decreased the pore volume and increased the pore size with a decrease on the surface area of about 60-75%. The matrices for enzyme immobilization were chosen considering the best combination of high surface area and hardness. Thus, the POS-PVA prepared with 5.56 x 10(-5) M of PVA with a surface area of 123 m(2)/g and hardness of 71 HV (50 gf 30 s) was shown to be suitable to immobilize the lipase, with an immobilization yield of about 40%. (c) 2008 Elsevier B.V. All rights reserved.

Influence of gelation time on the morphological and physico-chemical properties of the sol-gel entrapped lipase

Different gelation times (4, 18, 24 and 48 h) were used for the preparation of silica sol-gel supports and encapsulated Candida rugosa lipase using tetraethoxysilane (TEOS) as precursor. The hydrophobic matrices and immobilized lipases produced were characterized with regard to pore volume and size by nitrogen adsorption (BJH method), weight loss upon heating (TGA), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), chemical composition (FTIR) and percentage of hydrolysis (POH%) of olive oil. These structural parameters were found to change with the gelation time, but no direct relation was found between the percentage of oil hydrolysis (POH%) and the gelation time. The best combination of high thermal stability and high POH% (99.5%) occurred for encapsulated lipase produced with 24 h gelation time. (C) 2007 Elsevier B.V. All rights reserved.

A comparison between neodymium doped alumina samples obtained by Pechini and sol-gel methods using thermo-stimulated luminescence and SEM

Neodymium doped and undoped aluminum oxide samples were obtained using two different techniques: Pechini and sol-gel. Fine grained powders were produced using both procedures, which were analyzed using Scanning Electron Microscopy (SEM) and Thermo-Stimulated Luminescence (TSL). Results showed that neodymium ions incorporation is responsible for the creation of two new TSL peaks (125 and 265 degrees C) and, also, for the enhancement of the intrinsic TSL peak at 190 degrees C. An explanation was proposed for these observations. SEM gave the dimensions of the clusters produced by each method, showing that those obtained by Pechini are smaller than the ones produced by sol-gel; it can also explain the higher emission supplied by the first one. (C) 2010 Elsevier B.V. All rights reserved.

TL and OSL properties of KAlSi(3)O(8):Mn, obtained by sol-gel process

Thermoluminescence (TL) and Optically Stimulated Luminescence (OSL) properties of KAlSi(3)O(8):Mn glasses obtained through the sol gel technique were investigated. Samples were obtained with five different molar concentrations of 0.25, 0.5, 1, 2 and 5 mol% of manganese. Transmission Electronic Microscopy (TEM) indicated the occurrence of nanoparticles composed by glass matrix elements with Mn. Best results for TL response were obtained with 0.5 mol% Mn doped sample, which exhibits a TL peak at 180 degrees C. The TL spectrum of this sample presents a broad emission band from 450 to 700 nm with a peak at 575 nm approximately. The emission band fits very well with the characteristic lines of the Mn(2+) emission features. According to this fact, the band at 410 nm can be ascribed to (6)A(1)(S) -> (4)A(1)(G), (4)E(G) transition, while the 545 nm band can be attributed to the superposition of the transitions (6)A(1)(S) -> (4)T(2)(G) and (6)A(1)(S) -> (4)T(1)(G). The dependence of the TL response with the energy of X-rays (27-41 keV) showed a small decrease of the TL intensity in the high energy region. Excitation with blue LEDs showed OSL in the UV region with a fast decay component. (C) 2011 Elsevier Ltd. All rights reserved.

Erbium-activated silica-zirconia planar waveguides prepared by sol-gel route

Er(3+) doped (100-x)SiO(2)-xZrO(2) planar waveguides were prepared by the sol-gel route, with x ranging from 10 up to 30 mol%. Multilayer films doped with 0.3 mol% Er(3+) ions were deposited on fused quartz substrates by the dip-coating technique. The thickness and refractive index were measured by m-line spectroscopy at different wavelengths. The fabrication protocol was optimized in order to confine one propagating mode at 1.5 mu m. Photoluminescence in the near and visible region indicated a crystalline local environment for the Er(3+) ion. (c) 2007 Elsevier B.V. All rights reserved.

Sol-gel preparation of near-infrared broadband emitting Er(3+)-doped SiO(2)-Ta(2)O(5) nanocomposite films

This article reports a study on the preparation, densification process, and structural and optical properties of SiO(2)-Ta(2)O(5) nanocomposite films obtained by the sol-gel process. The films were doped with Er(3+) and the Si:Ta molar ratio was 90:10. Values of refractive index, thickness and vibrational modes in terms of the number of layers and thermal annealing time are described for the films. The densification process is accompanied by OH group elimination, increase in the refractive index, and changes in film thickness. Full densification of the film is acquired after 90 min of annealing at 900 degrees C. The onset of crystallization and devitrification, with the growth of Ta(2)O(5) nanocrystals occurs with film densification, evidenced by high-resolution transmission electron microscopy. The Er(3+)-doped nanocomposite annealed at 900 degrees C consists of Ta(2)O(5) nanoparticles, with sizes around 2 nm, dispersed in the SiO(2) amorphous phase. The main emission peak of the film is detected at around 1532 nm, which can be assigned to the (4)I(13/2)->(4)I(15/2) transition of the Er(3+) ions present in the nanocomposites. This band has a full width at half medium of 64 nm, and the lifetime measured for the (4)I(13/2) levels is 5.4 ms, which is broader compared to those of other silicate systems. In conclusion, the films obtained in this work are excellent candidates for use as active planar waveguide. (C) 2010 Elsevier B.V. All rights reserved.

Effect of mesoporosity of vanadium oxide prepared by sol-gel process as cathodic material evaluated by cyclability during Li(+) insertion/deinsertion

The effect of pore structure on the behavior of lithium intercalation into an electrode containing porous V(2)O(5) film has been investigated and compared with the electrode containing a non-porous V(2)O(5) film. X-ray diffraction patterns indicate a lamellar structure for both materials. Nitrogen adsorption isotherms, t-plot method, and Scanning Electronic Microscopy show that the route employed for the preparation of mesoporous V(2)O(5) was successful. The electrochemical performance of these matrices as lithium intercalation cathode materials was evaluated. The porous material reaches stability after several cycles more easily compared with the V(2)O(5) xerogel. Lithium intercalation into the porous V(2)O(5) film electrode is crucially influenced by pore surface and film surface irregularity, in contrast with the non-porous surface of the V(2)O(5) xerogel.

Novel molecular sieve silica (MSS) membranes: characterisation and permeation of single-step and two-step sol-gel membranes

High quality MSS membranes were synthesised by a single-step and two-step catalysed hydrolyses employing tetraethylorthosilicate (TEOS), absolute ethanol (EtOH), I M nitric acid (HNO3) and distilled water (H2O). The Si-29 NMR results showed that the two-step xerogels consistently had more contribution of silanol groups (Q(3) and Q(2)) than the single-step xerogel. According to the fractal theory, high contribution of Q(2) and Q(3) species are responsible for the formation of weakly branched systems leading to low pore volume of microporous dimension. The transport of diffusing gases in these membranes is shown to be activated as the permeance increased with temperature. Albeit the permeance of He for both single-step and two-step membranes are very similar, the two-step membranes permselectivity (ideal separation factor) for He/CO2 (69-319) and He/CH4 (585-958) are one to two orders of magnitude higher than the single-step membranes results of 2-7 and 69, respectively. The two-step membranes have high activation energy for He and H-2 permeance, in excess of 16 kJ mol(-1). The mobility energy for He permeance is three to six-fold higher for the two-step than the single-step membranes. As the mobility energy is higher for small pores than large pores and coupled with the permselectivity results, the two-step catalysed hydrolysis sol-gel process resulted in the formation of pore sizes in the region of 3 Angstrom while the single-step process tended to produce slightly larger pores. (C) 2002 Elsevier Science B.V. All rights reserved.