Cellulase immobilized by sol–gel entrapment for efficient hydrolysis of cellulose

Cellulase from Trichoderma reesei (Celluclast 1.5 L, Novozyme) was immobilized by sol–gel encapsulation, using binary or ternary mixtures of tetramethoxysilane (TMOS) with alkyl- or aryl-substituted trimethoxysilanes as precursors. Optimization of immobilization conditions resulted in 92 % recovery of total enzymatic activity in the best immobilized preparate. The immobilized cellulase exhibiting the highest activity, obtained from tetramethoxysilane and methyltrimethoxysilane precursors at 3:1 molar ratio, was investigated in the hydrolysis reaction of microcrystalline cellulose (Avicel PH101). Although the optimal values did not change significantly, both temperature and pH stabilities of the sol–gel entrapped cellulase improved compared to the native enzyme. Immobilization also conferred superior resistance against the inactivation effect of glucose. Reuse of the sol–gel entrapped cellulase showed 40 % retention of the initial activity after five batch hydrolysis cycles, demonstrating the potential of this biocatalyst for large-scale application.     

Effect of sol–gel encapsulation on the unfolding of ferric horse heart cytochrome c

Electronic absorption and resonance Raman spectra of ferric cytochrome c embedded in wet silica gels, in the presence of guanidine HCl as unfolding agent, between pH 0.35 and 7.0 are presented. The data clearly show that the ferric form of the protein encapsulated in sol–gel preserves its active site conformation. However, the spectra of the unfolded embedded protein are different from the corresponding spectra in solution suggesting that a strong interaction between the protein and the sol–gel takes place upon unfolding. The unfolding process mainly depends on the interaction between the exposed positive charges of the unfolded protein and the negatively charged functional groups of the silica surfaces. While this interaction partially stabilizes the protein in its native structure even at very acidic pH, in the presence of denaturants it has the opposite effect, causing mainly the weakening of both the heme-protein and the heme-ligand interactions.     

Improvement of catalytic properties of lipase from Arthrobacter sp. by encapsulation in hydrophobic sol–gel materials

In this work, lipase from Arthrobacter sp. was immobilized by sol–gel encapsulation to improve its catalytic properties. Various silanizing agents including vinyl-trimethoxy silane, octyl-trimethoxy silane, γ-(methacryloxypropyl)-trimethoxy silane (MAPTMS) and tetraethoxysilane (TEOS) were chosen as the precursors. Among them, MAPTMS was for the first time utilized to encapsulate lipases, and the prepared enzyme by copolymerization of MAPTMS and TEOS exhibited the highest activity in both the hydrolysis of p-nitrophenyl palmitate and the asymmetric acylation of 4-hydroxy-3-methyl-2-(2-propenyl)-2-cyclopenten-1-one. The effects of various immobilization parameters were investigated. Under the optimum conditions of MAPTMS/TEOS = 1/1 (mol/mol), water/silane molar ratio (R value) = 20 and lipase loading = 0.01 g/mL sol, the total activity of the immobilized enzyme reached up to 13.6-fold of the free form. Moreover, the encapsulated lipase exhibited higher thermal stability than the free form and retained 54% of the original activity after uses for 60 d. Enantioselectivity of enzyme was also improved with an E value of 150 after encapsulation from 85 for the free form.     

Kinetic study of the colloidal and enzymatic stability of β-galactosidase,for designing its encapsulation route through sol–gel route assisted by Triton X-100 surfactant

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.     

An innovative sol–gel naringinase bioencapsulation process for glycosides hydrolysis

In this work, a biocatalytic system was chosen to enable the deglycosylation of natural glycosides in order to improve their biological activity. Naringinase, which has both alpha-l-rhamnosidase and beta-d-glucosidase activities, was the enzyme used. Naringin, the glycoside substrate used in this bioconversion, and its aglycone product naringenin are compounds with interesting pharmacological activities such as anti-oxidant and anti-inflammatory; naringenin also acts as an anti-carcinogenic and neuroprotective agent, demonstrating a high potential for use by the pharmaceutical industry.Sol–gel, an innovative technique performed in aqueous media, was developed for naringinase immobilization in lens-shaped particles. Different sol–gel precursors, including tetramethoxysilane (TMOS), methyltrimethoxysilane (MTS), 3-aminopropyltrimethoxysilane (APS) and diglycerylsilane (DGS) with different aging times were tested in five consecutive trials. The best results were obtained with TMOS and TMOS/DGS after 4 h aging time and TMOS/glycerol and DGS at 14 h aging time. The characterization of these matrices was performed with respect to their diameter, volume, naringin and naringenin partition coefficients, and the optimum temperature and pH for naringinase activity. The operational stability of the bioencapsulated naringinase in the selected sol–gel matrices was studied through successive reutilizations. The naringinase sol–gel matrix (TMOS, DGS and TMOS/DGS) deactivation studies followed the Sadana model. The matrix TMOS/glycerol showed a constant 100% residual activity after 50 consecutive runs.     

Enhancement of enzymatic hydrolysis rate of olive oil in water by dimethyl β-cyclodextrin

Summary The hydrolysis rate of olive oil byCandida cylindracea lipase in an aqueous solution without surfactants can be increased up to 6-fold by the addition of up to 60 mg/ml of dimethyl -cyclodextrin (DMCD).     

Effect of visible light on catalytic hydrolysis of p-nitrophenyl palmitate by the Pseudomonas cepacia lipase immobilized on sol–gel support

This paper demonstrates Pseudomonas cepacia lipase catalyzed hydrolysis of p-nitrophenyl palmitate under irradiation of light with wavelengths of 250–750 nm. The reaction follows Michaelis–Menten Kinetics and the light irradiation increases the overall rate of hydrolysis. Using Lineweaver–Burk plot K _M and V _max values for the reaction in presence of light are found to be 39.07 and 66.67 mM/min/g, respectively; while for the same reaction under dark condition, the values are 7.08 and 10.21 mM/min/g. The linear form of enzyme dependent rate of reaction confirms that no mass-transfer limitations are present and the reaction is a kinetically controlled enzymatic reaction.     

Decolorization of azo dye by immobilized Pseudomonas luteola entrapped in alginate–silicate sol–gel beads

Pseudomonas luteola was immobilized by entrapment in alginate–silicate sol–gel beads for decolorization of the azo dye, Reactive Red 22. The influences of biomass loading and operating conditions on specific decolorization rate and dye removal efficiency were studied in details. The immobilized cells were found to be less sensitive to changes in agitation rates (dissolved oxygen levels) and pH values. Michaelis–Menten kinetics could be used to describe the decolorization kinetics with the kinetic parameters being 36.5 mg g⁻¹ h⁻¹, 300.1 mg l⁻¹ and 18.2 mg g⁻¹ h⁻¹, 449.8 mg l⁻¹ for free and immobilized cells, respectively. After five repeated batch cycles, the decolorization rate of the free cells decreased by nearly 54%, while immobilized cells still retained 82% of their original activity. The immobilized cells exhibited better thermal stability during storage and reaction when compared with free cells. From SEM observation, a dense silicate gel layer was found to surround the macroporous alginate–silicate core, which resulted in much improved mechanical stability over that of alginate beads when tested under shaking conditions. Alginate–silicate matrices appeared to be the best matrix for immobilization of P. luteola in decolorization of Reactive Red 22 when compared with previous results using synthetic or natural polymer matrices.     

Optimization of integrated alkaline–extrusion pretreatment of barley straw for sugar production by enzymatic hydrolysis

In this work, an integrated one-step alkaline–extrusion process was tested as pretreatment for sugar production from barley straw (BS) biomass. The influence of extrusion temperature (T) and the ratio NaOH/BS dry matter (w/w) (R) into the extruder on pretreatment effectiveness was investigated in a twin-screw extruder at bench scale. A 2³ factorial design of experiments was used to analyze the effect of process conditions [T: 50–100 °C; R: 2.5–7.5% (w/w)] on composition and enzymatic digestibility of pretreated substrate (extrudate). The optimum conditions for a maximum glucan to glucose conversion were determined to be R = 6% and T = 68 °C. At these conditions, glucan yield reached close to 90% of theoretical, while xylan conversion was 71% of theoretical. These values are 5 and 9 times higher than that of the untreated material, which supports the great potential of this one-step combined pre-treatment technology for sugar production from lignocellulosic substrates. The absence of sugar degradation products is a relevant advantage over other traditional methods for a biomass to ethanol production process since inhibitory effect of such product on sugar fermentation would be prevented.     

A model for the rate of enzymatic hydrolysis of cellulose in heterogeneous solid–liquid systems

Hydrolysis of cellulose by cellulase enzymes has been studied in a stirred batch reactor at 50°C. A kinetic model has been devised by which the behaviour of such a reaction could be described. The model has been developed on the basis of shrinking particle theory and Langmuir isotherm concept. The applicability of the model has been tested by comparing the experimental results for diverse reaction systems, obtained in the present study or taken from the literature, and those predicted from the model. The degree of agreement was within ±2–11%.     

Direct electrochemical immunoassay based on a silica nanoparticles/sol–gel composite architecture for encapsulation of immunoconjugate

A highly hydrophobic and non-toxic colloidal silica nanoparticle/polyvinyl butyral sol–gel composite membrane was prepared on a platinum wire electrode. With diphtheria-toxoid (D-Ag) as a model antigen and encapsulation of diphtheria antibody (D-Ab) in the composite architecture, this membrane could be used for reagentless electrochemical immunoassay. It displayed a porous and homogeneous composite architecture without the aggregation of the immobilized protein molecules. The formation of immunoconjugate by a simple one-step immunoreaction between D-Ag in sample solution and the immobilized D-Ab introduced the change in the potential. Under optimal conditions, the D-Ag analyte could be determined in the linear ranges from 10 to 800 ng ml⁻¹ with a relatively low detection limit of 2.3 ng ml⁻¹ at 3δ. The D-Ag immunosensor exhibited good precision, high sensitivity, acceptable stability, accuracy, and reproducibility. This composite membrane could be used efficiently for the entrapment of different biomarkers and clinical applications.     

Cryopreservation of in vitro-grown shoot tips of raspberry (Rubus idaeus L.) by encapsulation–vitrification and encapsulation–dehydration

The first efficient cryopreservation procedure for in vitro-grown shoot tips of raspberry (Rubus idaeus L.) has been developed based on encapsulation–vitrification (EnVi) and encapsulation–dehydration (EnDe). EnVi resulted in higher survival (85%) and regrowth (75%) of cryopreserved shoot tips than EnDe (65 and 50%, respectively). In both cryogenic procedures, shoots regenerated from cryopreserved shoot tips without intermediary callus formation. Histological studies showed that a much larger number of meristematic cells survived following EnVi than EnDe. The EnVi procedure was applied to seven raspberry genotypes with an average survival and regrowth of 71 and 68%, respectively. Regenerated plants showed normal morphology. Results here indicate EnVi as a simple and efficient method for long-term preservation of R. idaeus germplasm.     

Hydration dependent dynamics in sol–gel encapsulated myoglobin

In this work we study the effect of hydration on the dynamics of a protein in confined geometry, i.e. encapsulated in a porous silica matrix. Using elastic neutron scattering we investigate the temperature dependence of the mean square displacements of non-exchangeable hydrogen atoms of sol-gel encapsulated met-myoglobin. The study is extended to samples at 0.2, 0.3 and 0.5 g water/g protein fractions and comparison is made with met-myoglobin powders at the same average hydration and with a dry powder sample. Elastic data are analysed using a model of dynamical heterogeneity to take into account deviations of elastic intensity from gaussian behaviour in a large momentum transfer range and reveal a specific, model independent, effect of sol-gel confinement on protein dynamics, consisting mainly in a reduction of large-scale motions that are activated at temperatures larger than approximately 230 K. Surprisingly, the effect of confinement depends markedly on hydration and has a maximum at about 35% water/protein fraction corresponding to full first shell hydration. The presence of hydration-dependent MSD also in encapsulated met-Mb strongly supports the idea that the effect of sol-gel confinement on protein dynamics involves a modification of the structural/dynamical properties of the co-encapsulated solvent more than direct protein-matrix interactions.     

Sol–gel encapsulation of pullulanase in the presence of hybrid magnetic (Fe3O4–chitosan) nanoparticles improves thermal and operational stability

Bioprocess and Biosystems Engineering - Pullulanase was sol–gel encapsulated in the presence of magnetic chitosan/Fe3O4 nanoparticles. The resulting immobilized pullulanase was characterized...     

Encapsulation in a sol–gel matrix of lipase from Aspergillus niger obtained by bioconversion of a novel agricultural residue

Lipase from Aspergillus niger was obtained from the solid-state fermentation of a novel agroindustrial residue, pumpkin seed flour. The partially purified enzyme was encapsulated in a sol–gel matrix, resulting in an immobilization yield of 71.4 %. The optimum pH levels of the free and encapsulated enzymes were 4.0 and 3.0, respectively. The encapsulated enzyme showed greater thermal stability at temperatures of 45 and 60 °C than the free enzyme. The positive influence of the encapsulation process was observed on the thermal stability of the enzyme, since a longer half-life t _1/2 and lower deactivation constant were obtained with the encapsulated lipase when compared with the free lipase. Kinetic parameters were found to follow the Michaelis–Menten equation. The K _m values indicated that the encapsulation process reduced enzyme–substrate affinity and the V _max was about 31.3 % lower than that obtained with the free lipase. The operational stability was investigated, showing 50 % relative activity up to six cycles of reuse at pH 3.0 at 37 °C. Nevertheless, the production of lipase from agroindustrial residue associated with an efficient immobilization method, which promotes good catalytic properties of the enzyme, makes the process economically viable for future industrial applications.     

Immobilization of bacteria in silica matrices using citric acid in the sol–gel process

The aim of this work was to use citric acid in the sol–gel process to generate an inorganic polymer that allows bacterial survival for long periods of time and to study the influence of different storage temperatures. We compared gram-negative Escherichia coli and gram-positive Staphylococcus aureus, immobilized and preserved at different storage temperatures in silica matrices prepared by the method proposed. Immobilized E. coli and S. aureus in silica matrices were stored in sealed tubes at 20, 4, −20, and −70°C for 4 months during which the number of viable cells was analyzed. Results show that the immobilization in silica matrices using citric acid, to neutralize the alkalinity of the silica precursors, makes the technique not only biocompatible but also easier to perform since polymerization does not occur immediately as it does when hydrochloric acid is utilized.     

Determination of deoxynivalenol in organic and conventional food and feed by sol–gel immunoaffinity chromatography and HPLC–UV detection

The paper describes the determination of deoxynivalenol (DON) in 55 wheat food and feed samples, 26 from conventional and 29 from organic production. Immunoaffinity columns prepared by entrapping anti-DON antibodies by the sol–gel method were used for sample clean-up. DON was quantified by high performance liquid chromatography (HPLC) and ultraviolet (UV) detection. In general, the incidence of DON contamination was rather low. In eight samples (14.5%) the DON concentration was above the LOQ (380 ng/g), in six samples (10.9%) DON was detected but could not be quantified (>LOD (200 ng/g), <LOQ). In seven conventional samples (two pasta, two cookie, two snack and one feed sample) but only in one organic sample (a snack) the DON concentration was >LOQ. The data indicate both a higher incidence of DON contamination and higher DON concentrations in food and feed samples from conventional than in those from organic production.     

Determination of glassy state by cryo-SEM and DSC in cryopreservation of mint shoot tips by encapsulation–dehydration

Cryopreservation of mint shoot tips grown in vitro (Mentha × piperita) was performed after encapsulation in alginate beads. Encapsulated shoot tips were first precultured in sucrose enriched medium (0.75 M) and then dried under a sterile air flow (0–6 h). After cooling in liquid nitrogen and warming in a warm water bath, alginate beads were transferred to solid culture medium for 4 weeks. The effect of dehydration time of the encapsulated shoots was evaluated for water content, cooling and warming rates, ice crystal formation and cellular vitrification, by using low temperature scanning electron microscopy and differential scanning calorimetry. Viability of the recovered material showed a close relation between the dehydration time, cooling and warming rates, ice formation avoidance and tissue vitrification. At short drying periods (up to 3 h), ice crystals were formed and the viability was low or absent. After longer drying periods (5 and 6 h), both beads and specimens became vitrified.     

Photoluminescence and thermoluminescence properties of pure and rare-earth-doped ZrO2 prepared by Pechini-type sol–gel

In this article, photoluminescence (PL) and thermoluminescence (TL) properties of ZrO₂, ZrO₂:Dy³⁺, ZrO₂:Dy³⁺–Gd³⁺, ZrO₂:Dy³⁺–Yb³⁺, ZrO₂:Dy³⁺–Er³⁺, and ZrO₂:Dy³⁺–Sm³⁺ phosphors synthesized by the Pechini method were investigated. The crystal structure, thermal properties, morphology, PL and TL properties were investigated using X-ray powder diffraction (XRD), differential thermal analysis/thermogravimetric analysis (DTA/TGA), scanning electron microscopy (SEM), PL and TL, respectively. The room temperature emission bands corresponding to ⁴F_9/2 → ⁶H_J (J = 9/2, 11/2, 13/2 and 15/2) transitions of Dy³⁺ ions were measured. The phosphors were analysed using T_m–T_STOP, variable dose, and computerized glow curve fitting methods. Reusability, dose–response, and fading characteristics were investigated. The phosphors have a natural TL emission that vanished by heating treatment. Moreover, new peaks with similar properties to the natural emissions were observed after high-dose irradiation and long-term fading experiments. The glow curves of the phosphors have 13 individual peaks and many low- and high-temperature satellite peaks. The origin of the peaks is ZrO₂ host material and doping with rare-earth ions (Gd³⁺, Dy³⁺, Yb³⁺, Er³⁺ and Sm³⁺) does not lead to a new glow peak. The dopants cause drastic changes in individual peak intensities of ZrO₂.The initial fading rates of all the phosphors are relatively fast, but they slow down as time goes on.