Controlled Pore Silica Research Articles

Immobilization of commercial cyclomaltodextrin glucanotransferase into controlled pore silica by the anchorage method and covalent bonding

Abstract Cyclomaltodextrin glucanotransferase (CGTase) is used mainly for the industrial production of cyclodextrins (CDs). Their potential use and availability have played a decisive role in increasing demand for CDs. The aim of the present study was to immobilize the commercial CGTase (Toruzyme® 3.0L) into controlled pore silica by surface anchoring (SCGT-A) and covalent bonding (SCGT-CB) and to apply the immobilized enzymes in the production of CDs. The SCGT-A method proved to be efficient due to its short immobilization time and, it was possible to immobilize 89.63% of CGTase, in terms of activity. Commercial CGTase immobilized by SCGT-CB provided an immobilization yield of 48.44%, also in terms of activity. There was an increase in total CD production of 60.97% and 34.90% for SCGT-A and SCGT-CB, respectively, in a reaction medium with 10% ethanol, compared to the same media without ethanol. Higher CD production occured at 24 and 48 h for SCGT-A and SCGT-CB methods, respectively. The use of SCGT-A was more efficient than SCGT-CB and free enzyme (CGT-F) after 24 h of reaction. The increase production of CDs obtained by the SCGT-A method and the demonstrated continuous production of CDs with the SCGT-CB method make the use of immobilized CGTase viable for industrial applications.

Silica glass modified with flavonoid derivatives for preconcentration of some toxic metal ions in water samples and their determination with ICP-MS

Quercetin (3,3',4',5,7-pentahydroxyflavone) chemically bonded through pyran rearrangement on modified controlled pore silica glass (QCPSG) with a capacity 0.213 mmol/g was used for solid phase extraction of some toxic metal and metalloid ions. The newly designed QCPSG quantitatively sorbs As(V), Cd(II), Hg(II), and Pb(II) at the pH range 7.5-8.5 after 10 min of stirring. HCl (1 mol L(-1)) instantaneously elutes all the metal ions. The sorption capacity of the ion collector is 0.42, 0.46, 0.53, and 0.49 mmol g(-1) for As(V), Cd(II), Hg(II), and Pb(II), respectively, whereas the preconcentration factor is 200. The effect of NaCl, Na2SO4, NaF, NaBr, Na3PO4, and other interfering salts on the sorption of metal ions (50 microg L(-1)) was reported. Analytical detection limits of As(V), Cd(II), Hg(II), and Pb(II) were 4.18, 2.44, 15.86, and 25.00 pg mL(-1), respectively. QCPSG was used in the separation of the investigated metal ions from some natural water samples collected from diverse origins followed by determination by inductively coupled plasma-mass spectrometry. The data were compared with those obtained by the standard methods of determination using atomic absorption (hydride generation, HGAAS and after solvent extraction with ammonium pyrolidine dithiocarbamate/methyl isobutyl ketone). The suggested solid phase extraction method was found accurate with no random error.

Crossover from Normal to Inverse Temperature Dependence in the Adsorption of Nonionic Surfactants at Hydrophilic Surfaces and Pore Walls

The adsorption of the nonionic surfactant C8E4 from its aqueous solutions to the pore walls of four controlled-pore silica glass (CPG) materials of different mean pore widths (10−50 nm) has been studied in a temperature range from 5 to 45 °C, that is, close to the lower critical temperature of liquid−liquid phase separation of the bulk system (Tc ≈ 40 °C). Pronounced S-shaped isotherms, with a normal temperature dependence of the adsorption in the initial low-affinity region but an inverse temperature dependence in the plateau region, are found with all CPG materials. The experimental adsorption isotherms are compared with predictions of a theoretical model (Phys. Rev. Lett. 2004, 92, 135701), which takes into account H-bonding and micelle formation in the bulk and at the surface. It is found that this model reproduces all peculiarities of the adsorption in the present systems. The following conclusions emerge from this analysis: (1) At low bulk concentrations, the surfactant is adsorbed only in monomeric form (Henry's law behavior). In this regime, there is an energetic driving force for surfactant adsorption (in spite of the fact that nonselective pore walls are assumed), in agreement with the observed normal temperature dependence of the adsorption in this regime. (2) Surface aggregation observed at higher concentrations involves an energy penalty due to the loss of H bonds, which is overcompensated by the gain of (rotational) entropy of the H-bonding sites of the surfactant heads. Accordingly, surface aggregation is entropy-driven and endothermic and thus shows inverse temperature dependence. Hence, the model accounts for the observed crossover in the temperature dependence of the adsorption at the critical surface aggregation concentration.

Lipase‐catalyzed synthesis of butyl esters by direct esterification in solvent‐free system

AbstractBACKGROUND:Reactions performed under solvent‐free conditions give processes that are environmentally friendly, since most solvents are polluting agents. In this work, the performance of Candida rugosa lipae (CRL) immobilized on styrene‐divinylbenzene (STY‐DVB) or controlled pore silica (CPS), and the commercial lipase Novozym 435, was evaluated for the synthesis of butyl esters in solvent–free systems (SFS). A 22 full factorial design was used to study the influence of the organic acid chain length and the biocatalyst concentration on the esterification performance.RESULTS:When CRL on STY‐DVB was used, the ester formation was influenced by both variables and their interaction. The reaction conversion was higher (63%) using 10% of immobilized system and lauric acid, corresponding to a productivity of 3.62 g L−1 h−1 For CRL on CPS, only the effect of biocatalyst concentration was significant, and the highest yield was attained using 20% of immobilized system and caprilic acid. In the case of Novozym 435, the highest yield (49%) was obtained using butyric acid as acyl donor at 15% of immobilized lipase.CONCLUSION:The results allowed better understanding of the influence of important parameters in this environmentally friendly process, which also has the process advantage of a higher volumetric productivity when compared with a solvent system. Copyright © 2007 Society of Chemical Industry

Atividade e estabilidade operacional de lipase imobilizada em fosfato de zircônio na ausência e presença de polietilenoglicol

In the present work, different immobilization procedures were evaluated: direct addition of the lipase on zirconium phosphate (ZrP), and simultaneous addition of lipase and stabilizing agent (polyethylene glycol, MM-1500) on the same support. The selection of this additive was based on its compatibility with the lipase from Candida rugosa according to data previously obtained using controlled pore silica as support. The methodology of lipase immobilization on ZrP in the presence of PEG-1500 supplied more active immobilized derivatives (310.6 U mg -1 ) when compared with those obtained without addition of PEG-1500 (162.8 U mg -1 ), providing high activity recovery in the immobilization step (88.7%). Better performance of the immobilized derivative prepared in the presence of PEG was also verified in organic medium (synthesis of butyl butyrate), in terms of activity and operational stability. These results can be attributed to the enzyme coated by the additive that probably increased the access of the substrate to the active site of the lipase

Optimised immobilisation of microbial lipase on hydrous niobium oxide

AbstractThe aim of this work was to establish appropriate conditions for immobilising Candida rugosa lipase on a low‐cost inorganic matrix, hydrous niobium oxide, using a multivariate statistical approach. A 23 full factorial design was employed to determine the effects of support activation with glutaraldehyde (concentration 2.5–4.5%, pH 7–10) and lipase loading (200‐700 U g−1 matrix) on the hydrolytic and synthetic activities of the immobilised derivatives. From the results the following conditions were established: lipase loading of 450 U g−1 matrix and niobium oxide activation with glutaraldehyde at a concentration of 2.5% and pH 8. Under these conditions, high activity recovery (47.21%) and esterification yield (86.90%) were attained. The results also show that hydrous niobium oxide can be a valid alternative to replace high‐cost, commercially available inorganic matrices such as controlled pore silica. Copyright © 2006 Society of Chemical Industry

Local structure of a phase-separating binary mixture in a mesoporous glass matrix studied by small-angle neutron scattering

The mesoscopic structure of the binary system isobutyric acid + heavy water (D(2)O) confined in a porous glass (controlled-pore silica glass, mean pore width ca. 10 nm) was studied by small-angle neutron scattering at off-critical compositions in a temperature range above and below the upper critical solution point. The scattering data were analyzed in terms of a structure factor model similar to that proposed by Formisano and Teixeira [Eur. Phys. J. E 1, 1 (2000)], but allowing for both Ornstein-Zernike-type composition fluctuations and domainlike structures in the microphase-separated state of the pore liquid. The results indicate that the phase separation in the pores is shifted by ca. 10 K and spread out in temperature. Microphase separation is pictured as a transition from partial segregation at high temperature, due to the strong preferential adsorption of water at the pore wall, to a tube or capsule configuration of the two phases at low temperatures, depending on the overall composition of the pore liquid. Results for samples in which the composition of the pore liquid can vary with temperature due to equilibration with extra-pore liquid are consistent with this picture.

Efeito do polietilenoglicol e da albumina na imobilização de lipase microbiana e na catálise em meio orgânico

Poly (ethylene) glycol (PEG) and bovine serum albumin (BSA), as additive agents, were used to enhance the activity of immobilized microbial lipase in organic solvent. Controlled pore silica (CPS) was selected as matrix and different immobilization procedures were evaluated: directly lipase binding on CPS and simultaneous addition of lipase and additive agent on the same support. The highest coupling yield (59.6%) was attained when the immobilization procedure was performed at lipase loading of 150 U/g support in the presence of PEG-1.500. This immobilized system was used in esterification reactions under repeated batch cycles and the biocatalyst half-life was found to increase 2.7 times when compared with the control.

Controlled-pore silica glass modified with N-propylsalicylaldimine for the separation and preconcentration of trace Al(III), Ag(I) and Hg(II) in water samples.

Controlled-pore silica glass modified with N-propylsalicylaldimine (SCPSG) has been investigated as a surface-active matrix for the separation of some metal ions. The porous silica glass base was confirmed to have better stability towards hydrolysis in aqueous solution buffered at pH=9 in comparison to silica gel, which showed twice the surface area of controlled-pore silica glass. The different analytical parameters affecting the batch mode separation and preconcentration of trace Al(II), Ag(I) and Hg(II) in environmental samples using SCPSG, prior to their determination using inductively coupled plasma mass spectrometry (ICP-MS), were studied. The optimum conditions are pH 9.0 +/- 0.1, time of stirring 30 min and the eluent concentration 0.5 mol dm(-3) HNO3. The ion-exchange capacity of SCPSG with respect to Al(III), Ag(I) and Hg(II) was 0.27, 0.18 and 0.23 mmol g(-1), respectively. The recovery values for the metal ions were 96.8 +/- 0.86, 98.1 +/- 0.60 and 96.2 +/- 1.06%, and the analytical detection limits were 26.1, 1.49 and 0.44 pg cm(-3), respectively, for a preconcentration factor of 100. The method has been applied to the determination of the investigated metal ions in natural water samples as well as certified and reported samples and the results were found to be accurate.

Chemical modification of controlled‐pore silica with N‐propylsalicylaldimine

AbstractTwo controlled‐pore silica phases were prepared with a sol–gel precursor from a sodium silicate solution. N‐Propylsalicylaldimine was immobilized on these silica species to be used as chelating ion exchangers (IE11 and IE2). The monomer phase was also prepared for comparison. The N‐propylsalicylaldimine moiety was detected by Fourier transform infrared and ultraviolet in both the ion exchangers and the monomer phases. 1H‐NMR and mass spectrometry of the monomer also confirmed the structure. The capacity (C) of the ion exchangers was dependent on the porosity of the ion exchanger (CIE11 = 0.36 mmol of Cu/g and CIE2 = 0.026 mmol of Cu/g). The uptake behavior of IE11 toward some metal ions was studied, and log distribution coefficient (kd) was within the range of 2.19–5.16. Also, thermogravimetric and differential thermogravimetric analysis data were used to study the kinetics of the thermal decomposition process of IE11. Some thermodynamic parameters for the ion exchanger were calculated by the application of the rate theory of the first‐order reaction. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 88: 3159–3167, 2003

Flow injection spectrophotometric determination of isoproterenol using an avocado ( Persea americana) crude extract immobilized on controlled-pore silica reactor

An enzymatic reactor was constructed by the immobilization of polyphenol oxidase (PPO) from avocado ( Persea americana) crude extract in an inorganic support of controlled pore silica (CPS), after a previous step of silanization. This inorganic support has been used as an excellent carrier to immobilize this enzyme and the enzymatic reactor was used in a flow injection system for the determination of isoproterenol in pharmaceutical products. The procedure is based on the oxidation reaction of this drug with immobilized PPO and the product obtained was monitored at 492 nm. This system presented an analytical curve from 1.23×10 −4 to 7.38×10 −4 mol l −1 isoproterenol with a detection limit of 6.25×10 −5 mol l −1. Recoveries of isoproterenol between 98.5 and 103.1%, a relative standard deviation (R.S.D.) less than 1% ( n=10) and 36 determinations per h were obtained.

Selection of stabilizing additive for lipase immobilization on controlled pore silica by factorial design.

Candida rugosa lipase was covalently immobilized on silanized controlled pore silica (CPS) previously activated with glutaraldehyde in the presence of several additives to improve the performance of the immobilized form in long-term operation. Proteins (albumin and lecithin) and organic molecules (beta-cyclodextrin and polyethylene glycol [PEG]-1500) were added during the immobilization procedure, and their effects are reported and compared to the behavior of the immobilized biocatalyst in the absence (lacking) of additive. The selection of the most efficient additive at different lipase loadings (150-450 U/g of dry support) was performed by experimental design. Two 22 full factorial designs with two repetitions at the center point were employed to evaluate the immobilization yield. A better stabilizing effect was found when small amounts of albumin or PEG-1500 were added simultaneously to the lipase onto the support. The catalytic activity had a maximum (193 U/mg) for lipase loading of 150 U/g of dry support using PEG-1500 as the stabilizing additive. This immobilized system was used to perform esterification reactions under repeated batch cycles (for the synthesis of butyl butyrate as a model). The half-life of the lipase immobilized on CPS in the presence of PEG-1500 was found to increase fivefold compared with the control (immobilized lipase on CPS without additive).

Thermal stability and deactivation energy of free and immobilized invertase

The thermal stability and the energy of deactivation of free invertase and the immobilized enzyme (IE) was measured at temperatures in the range of 35 to 65°C for the hydrolysis of a 5% w/v sucrose solution. The free enzyme at pH 5.0 is stable up to 50°C for a period of 4 h. Invertase immobilized in controlled pore silica by the silane-glutaraldehyde covalent method is stable up to 55oC, in pH 4.5 for the same period. For higher temperatures the enzyme deactivation follows the exponential decay model and half-lives are 0.53, 1.80, and 13.9 h for free invertase, at 65, 60, and 55oC, respectively. For the IE half-lives are 0.48, 1.83, and 20.9 h, at 65, 60, and 55oC, respectively. The IE is more stable than the free invertase; the energy of deactivation being 83.1 kcal/mol for the IE and 72.0 kcal/mol for the free enzyme.

Thermal stability and energy of deactivation of free and immobilized cellobiase

Commercial cellobiase has been immobilized in controlled pore silica particles by covalent binding with the silane-glutaraldehyde method with protein and activity yields of 67% and 13.7%, respectively. Thermal stability of the free and immobilized enzyme (IE) was determined with 0.2% w/v cellobiose solution, pH 4.8, temperatures from 40 to 70°C for free enzyme and 40 to 75°C for IE. Free cellobiase maintained its activity practically constant for 240 min at temperatures up to 55°C. The IE has shown higher stability retaining its activity in the same test up to 60° C. Half-lives for free enzyme were 14.1, 2.1 and 0.17 h at 60, 65 and 70°C, respectively, whereas the IE at the same temperatures had half-lives of 245, 21.3 and 2.9 h. The energy of thermal deactivation was 80.6 kcal/mol for the free enzyme and 85.2 kcal/mol for the IE, confirming stabilization by immobilization.

Characterization of free and immobilized invertase regarding activity and energy of activation

Invertase from NOVO Nordisk has been immobilized in controlled pore silica particles (diameter: 0.351 mm and mean pore size: 37.5 nm) by covalent binding with the silane-glutaraldehyde method. The activity of the free and immobilized enzyme (IE) was determined with 5% (w/v) sucrose, at 35 to 65oC and pH from 3 to 7. Maximum activities were found in the pH range from 5 to 6 for free invertase, and pH 4.5 for the IE. Activity yield for the IE was 24%. The Energy of Activation (Ea) was found to be a function of pH, giving for free invertase, Ea = 7.0 and 6.86 kcal/mol at pH 5.0 and 5.5, respectively, whereas for the immobilized enzyme, Ea = 6.55 and 5.93 kcal/mol at pH 4.5 and 5.0, respectively.

Water dynamics in controlled pore silica glasses

Water in porous silica glass is a suitable system for investigating the effect of confinement on translational diffusion. These systems are important because of their relevance in catalytic and separation processes. Two factors are to be considered in the case of confined water: (1) the effects of hydrophilic and hydrophobic surfaces on interfacial water and (2) how the dynamics of the hydrogen bond network changes due to the volume of confinement. Here quasi-elastic neutron scattering experiments at room temperature on water filled controlled pore glasses with radius of 15, 24 and 32 {angstrom}, are presented and analyzed using the random-jump diffusion model. Both the average residence time and the mean jump distance increase with decreasing pore radius.

Production of cyclodextrins in a fluidized-bed reactor using cyclodextrin-glycosyl-transferase.

Cyclodextrin-glycosyl-transferase (EC2.4.1.19), produced by Wacker (Munich, Germany), was purified by biospecific affinity chromatography with beta-cyclodextrin (beta-CD) as ligand, and immobilized into controlled pore silica particles (0.42 mm). This immobilized enzyme (IE) had 4.7 mg of protein/g of support and a specific activity of 8.6 mumol of beta-CD/(min.gIE) at 50 degrees C, pH 8.0. It was used in a fluidized-bed reactor (FBR) at the same conditions for producing cyclodextrins (CDs) with 10% (w/v) maltodextrin solution as substrate. Bed expansion was modeled by the Richardson and Zaki equation, giving a good fit in two distinct ranges of bed porosities. The minimum fluidization velocity was 0.045 cm/s, the bed expansion coefficient was 3.98, and the particle terminal velocity was 2.4 cm/s. The FBR achieved high productivity, reaching in only 4 min of residence time the same amount of CDs normally achieved in a batch reactor with free enzyme after 24 h of reaction, namely, 10.4 mM beta-CD and 2.3 mM gamma-CD.

Characterization and utilization of Candida rugosa lipase immobilized on controlled pore silica.

Candida rugosa lipase was immobilized by covalent binding on controlled pore silica (CPS) using glutaraldehyde as cross-linking agent under aqueous and nonaqueous conditions. The immobilized C. rugosa was more active when the coupling procedure was performed in the presence of a nonpolar solvent, hexane. Similar optima pH (7.5-8.0) was found for both free and immobilized lipase. The optimum temperature for the immobilized lipase was about 10 degrees C higher than that for the free lipase. The thermal stability of the CPS lipase was also greater than the original lipase preparation. Studies on the operational stability of CPS lipase revealed good potential for recycling under aqueous (olive-oil hydrolysis) and nonaqueous (butyl butyrate synthesis) conditions.

Thermal stability and energy of deactivation of free and immobilized amyloglucosidase in the saccharification of liquefied cassava starch.

Amyloglucosidase from Novo (Copenhagen, Denmark) was immobilized in controlled pore silica particles with the silane-glutaraldehyde covalent method. Thermal stability of the free and immobilized enzyme (IE) was determined with 30% (w/v) alpha-amylase liquefied cassava starch, pH 4.5, temperatures from 35 to 75 degrees C. Free amyloglucosidase maintained its activity practically constant for 240 min and temperatures up to 50 degree C. The IE has shown higher stability retaining its activity for the same period up to 60 degrees C. Half-life for free enzyme was 20.6, 6.44, 2.07, 0.69, and 0.24 h for 55, 60, 65, 70, and 75 degrees C, respectively, whereas the IE at the same temperatures had half-lives of 116.4, 30.88, 8.52, 2.44, and 0.73 h. The energy of thermal deactivation was thus 50.6 and 57.6 kcal/mol, respectively for the free and IE, confirming stabilization by immobilization.

<FONT FACE=Symbol>b</font>-GALACTOSIDASE IMMOBILIZATION ON CONTROLLED PORE SILICA

The immobilization of b -galactosidase from Kluyveromyces fragilis on controlled pore silica was investigated. Immobilization was performed on amino silica activated with glutaraldehyde and the product was applied to the hydrolysis of lactose of whey. The behaviors of the soluble and immobilized enzyme were compared by using whey and a lactose solution as the substrate. With the aim of optimizing the method, parameters such as the amount of glutaraldehyde and the size of the particles were evaluated by comparing activities and stabilities on batch and continuously fluidized bed reactors