Affinity Covalent Immobilization of Glucoamylase onto ρ-Benzoquinone-Activated Alginate Beads: II. Enzyme Immobilization and Characterization

A novel affinity covalent immobilization technique of glucoamylase enzyme onto ρ-benzoquinone-activated alginate beads was presented and compared with traditional entrapment one. Factors affecting the immobilization process such as enzyme concentration, alginate concentration, calcium chloride concentration, cross-linking time, and temperature were studied. No shift in the optimum temperature and pH of immobilized enzymes was observed. In addition, K _m values of free and entrapped glucoamylase were found to be almost identical, while the covalently immobilized enzyme shows the lowest affinity for substrate. In accordance, V _m value of covalently immobilized enzyme was found lowest among free and immobilized counter parts. On the other hand, the retained activity of covalently immobilized glucoamylase has been improved and was found higher than that of entrapped one. Finally, the industrial applicability of covalently immobilized glucoamylase has been investigated through monitoring both shelf and operational stability characters. The covalently immobilized enzyme kept its activity over 36 days of shelf storage and after 30 repeated use runs. Drying the catalytic beads greatly reduced its activity in the beginning but recovered its lost part during use. In general, the newly developed affinity covalent immobilization technique of glucoamylase onto ρ-benzoquinone-activated alginate carrier is simple yet effective and could be used for the immobilization of some other enzymes especially amylases.     

Atrazine degradation by Pseudomonas strain ADP entrapped in sol-gel glass

Sol-gel entrapment was evaluated as a method for immobilization of an atrazine degrading Pseudomonas. It was found that the bacterium lost much of its atrazine degrading activity upon immobilization. However, partial activity could have been restored by amendment of nutrients. Bacteria immobilized using a prehydrolysis technique for the preparation of the sol-gel, retained better activity in comparison to bacteria immobilized using a composite calcium alginate/sol-gel procedure. Further study is underway to improve the activity of sol-gel entrapped bacteria.     

Fabrication of poly(ethylene glycol)‐based hydrogels entrapping enzyme‐immobilized silica nanoparticles

In this study, we immobilized enzymes by combining covalent surface immobilization and hydrogel entrapment. A model enzyme, glucose oxidase (GOX), was first covalently immobilized on the surface of silica nanoparticles (SNPs) via 3‐aminopropyltriethoxysilane (APTES), and the resultant SNP‐immobilized enzyme was physically entrapped within photopolymerized hydrogels prepared from two different molecular weights (MWs) (575 and 8000 Da) of poly(ethylene glycol)(PEG). The hydrogel entrapment resulted in a decrease in reaction rate and an increase in apparent K_m of SNP‐immobilized GOX, but these negative effects could be minimized by using hydrogel with a higher MW PEG, which provides higher water content and larger mesh size. The catalytic rate of the PEG 8000 hydrogel was about ten times faster than that of the PEG 575 hydrogel because of enhanced mass transfer. Long‐term stability test demonstrated that SNP‐immobilized GOX entrapped within hydrogel maintained more than 60% of its initial activity after a week, whereas non‐entrapped SNP‐immobilized GOX and entrapped GOX without SNP immobilization maintained less than 20% of their initial activity. Incorporation of SNPs into hydrogel enhanced the mechanical strength of the hydrogel six‐fold relative to bare hydrogels. Finally, a hydrogel microarray entrapping SNP‐immobilized GOX was fabricated using photolithography and successfully used for quantitative glucose detection. Copyright © 2009 John Wiley & Sons, Ltd.     

Immobilization of isolated and cellular hydrogenase ofD. desulfuricans in radiation polymerized polyacrylamides

Purified hydrogenase fromDesulfovibrio desulfuricans was immobilized either by entrapment or absorption onto porous neutral and charged acrylamide beads. Surface absorption and crosslinking on the beads resulted in a high hydrogenase activity and a good immobilization coefficient compared to the enzyme and whole cells entrapped in the same matrix. Maximum enzyme activity (citrate-phosphate buffer) was shifted to pH 6.5 upon immobilization in contrast to 6.0 for the free enzyme and the range of 6–7 for whole cells. Both the purified enzyme and whole cells were most active when held in neutral matrices. Immobilization improved the temperature stability (65‡C) and long term storage (4‡C) of the hydrogenase activity of both the purified enzyme and whole cells.     

Characteristics and antioxidant activity of catechin-loaded calcium pectinate gel beads prepared by internal gelation

Catechin-loaded calcium pectinate gel beads prepared by internal gelation were characterized for their catechin entrapment efficiency and release behavior. The entrapment efficiency was higher when the beads were prepared with a lower catechin-to-pectin ratio, shorter gelling time, higher pectin concentration, and lower acetic acid concentration. The entrapment efficiency was much higher under all tested conditions, when the beads were prepared by internal gelation instead of external gelation. The catechin release was slower for the beads prepared with lower catechin-to-pectin ratio, longer gelling time, and higher concentrations of pectin and acetic acid in both simulated gastric and intestinal fluids. Antioxidant power of catechin was effectively maintained in alkaline simulated intestinal fluid when catechin was entrapped within the beads, compared to cases where it was not entrapped, indicating that the beads can protect catechin molecules from the alkaline environment and release them in a sustained fashion.     

Effect of Preparation Conditions on the Stability of Pt/Al2O3 Catalysts in Methane Combustion

Alumina supported platinum catalysts were prepared by sol-gel method using aluminum-sec-butoxide, platinum acetylacetonate and sec-butanol. Various gelation process were performed either by the help of a gelation agent (water or acetic acid) or by a slow condensation without hydrolysis source. The textural and the structural study of the catalysts using the nitrogen physical adsorption and the hydrogen chemisorption showed that the BET surface area and the metal dispersion varied when the hydrolysis and the gelation processes were modified. The catalytic activities for methane combustion performed on the fresh and aged catalysts were correlated to the metal dispersion values. In addition the catalytic activity loss under reaction mixture seemed to be caused by the metal dispersion decrease. When the catalysts were aged under the reaction mixture at 600°C for 72 h, a BET surface area decrease and a metallic surface area loss were observed. The resistance to sintering observed to be dependant on the hydrolysis and the gelation processes was in favor of the catalyst prepared by acetic acid.     

Immobilized enzyme reactor chromatography: optimization of protein retention and enzyme activity in monolithic silica stationary phases

Our group recently reported on the application of protein-doped monolithic silica columns for immobilized enzyme reactor chromatography, which allowed screening of enzyme inhibitors present in mixtures using mass spectrometry for detection. The enzyme was immobilized by entrapment within a bimodal meso/macroporous silica material prepared by a biocompatible sol-gel processing route. While such columns proved to be useful for applications such as screening of protein-ligand interactions, significant amounts of entrapped proteins leached from the columns owing to the high proportion of macropores within the materials. Herein, we describe a detailed study of factors affecting the morphology of protein-doped bioaffinity columns and demonstrate that specific pH values and concentrations of poly(ethylene glycol) can be used to prepare essentially mesoporous columns that retain over 80% of initially loaded enzyme in an active and accessible form and yet still retain sufficient porosity to allow pressure-driven flow in the low μL/min range. Using the enzyme γ-glutamyl transpeptidase (γ-GT), we further evaluated the catalytic constants of the enzyme entrapped in capillary columns with different silica morphologies as a function of flowrate and backpressure using the enzyme reactor assay mode. It was found that the apparent activity of the enzyme was highest in mesoporous columns that retained high levels of enzyme. In such columns, enzyme activity increased by ∼2-fold with increases in both flowrate (from 250 to 1000 nL/min) and backpressure generated (from 500 to 2100 psi) during the chromatographic activity assay owing to increases in k_cat and decreases in K_M, switching from diffusion controlled to reaction controlled conditions at ca. 2000 psi. These results suggest that columns with minimal macropore volumes (<5%) are advantageous for the entrapment of soluble proteins for bioaffinity and bioreactor chromatography.     

Optimization of tetramethoxysilane-derived sol gel entrapment protocol stabilizes highly active chlorophyllase

Entrapment of membrane proteins is a challenging task compared to that involving soluble proteins. Chlorophyllase, a membrane protein, was successfully entrapped in tetramethoxysilane-derived sol-gel. Pre-gel sol typically consists of an aqueous suspension of chlorophyllase, precursors including tetramethoxysilane and/or methytrimethoxysilane, and sodium fluoride as catalyst. To obtain a highly active entrapped enzyme preparation, the effects of various immobilization parameters, including the chemical compositions of pre-gel sol (water/silane ratio, precursor type and proportions, enzyme loading, sodium fluoride concentration), and sol-gel process parameters (aging and drying time and approach) have been investigated. Chlorophyllase demonstrated the highest activity in gel derived from a pre-gel sol with water/silane ratio of 30 and enzyme loading of 0.257 mg_protein/g_gel, and showed moderately lower activity in organically modified sol-gel than that in hydrophilic sol-gel. The effects of water/silane ratio and precursor combinations on the activity of entrapped chlorophyllase were also studied by examining the pore morphology of gel via nitrogen adsorption-desorption. Longer aging time leads to an entrapped chlorophyllase preparation with higher activity. Chlorophyllase preparation demonstrated negligible activity after air-drying for 12 h while lyophilized chlorophyllase preparation demonstrated 8, 4 and 4 times higher activity than air-dried, vacuum-dried and solvent-dried preparations. Chlorophyllase demonstrated 30% higher activity in the improved sol-gel protocol than that from a non-optimized sol-gel protocol developed in a previous study.     

One-pot sequences of reactions with sol-gel entrapped opposing reagents: an enzyme and metal-complex catalysts

We extend our sol-gel methodology of one-pot sequences of reactions with opposing reagents to an enzyme/metal-complex pair. Sol-gel entrapped lipase and sol-gel entrapped RhCl[P(C(6)H(5))(3)](3) or Rh(2)Co(2)(CO)(12) were used for one-pot esterification and C-C double bond hydrogenation reactions, leading to saturated esters in good yields. When only the enzyme is entrapped, the homogeneous catalysts quench its activity and poison it. Thus, when 10-undecenoic acid and 1-pentanol were subjected in one pot to the entrapped lipase and to homogeneously dissolved RhCl[P(C(6)H(5))(3)](3) under hydrogen pressure, only 7% of the saturated 1-pentyl undecanoate was obtained. The yield jumped 6.5-fold when both the enzyme and the catalyst were immobilized separately in silica sol-gel matrixes. Similar one-pot esterifications and hydrogenations by sol-gel entrapped lipase and heterogenized rhodium complexes were carried out successfully with the saturated nonoic, undecanoic, and lauric acids together with several saturated and unsaturated alcohols. The use of (S)-(-)-2-methylbutanol afforded an optically pure ester. The heterogenized lipase is capable of inducing asymmetry during esterification with a prochiral alcohol. Both the entrapped lipase and the immobilized rhodium catalysts can be recovered simply by filtration and recycled in further runs without loss of catalytic activity.     

Stabilizing structure-switching signaling RNA aptamers by entrapment in sol-gel derived materials for solid-phase assays

Structure-switching, fluorescence-signaling DNA and RNA aptamers have been reported as highly versatile molecular recognition elements for biosensor development. While structure-switching DNA aptamers have been utilized for solid-phase sensing, equivalent RNA aptamers have yet to be successfully utilized in solid-phase sensors due to their lack of chemical stability and susceptibility to nuclease attack. In this study, we examined entrapment into sol-gel derived organic-inorganic composite materials as a platform for immobilization of structure-switching fluorescence-signaling RNA aptamer reporters, using both the synthetic theophylline- and naturally occurring thiamine pyrophosphate-binding RNA aptamers as test cases. Structure-switching versions of both aptamers were entrapped into a series of sol-gel derived composites, ranging from highly polar silica to hydrophobic methylsilsesquioxane-based materials, and the target-binding and signaling capabilities of these immobilized aptamers were assessed relative to solution. Both immobilized aptamers demonstrated sensitivity and selectivity similar to that of free aptamers when entrapped in a composite material derived from 40% (v/v) methyltrimethoxysilane/tetramethoxysilane. Importantly, this material also conferred protection from nuclease degradation and imparted long-term chemical stability to the RNA reporter systems. Given the versatility of sol-gel entrapment for development of biosensors, microarrays, bioaffinity columns, and other devices, this entrapment method should provide a useful platform for numerous solid-phase RNA aptamer-based devices.     

Thermally reversible polymer gels for biohybrid artificial pancreas

As an approach to replacing islets of Langerhans in an implanted biohybrid artificial pancreas, thermally reversible polymers based on N-isopropylacrylamide were synthesized and then evaluated as an extracellular matrix for islets in an immunoprotecting membrane pouch. A high molecular weight poly(N-isopropylacrylamide-co-acrylic acid (2 mole % in feed)) demonstrated gelation at 37°C and became a solution below 30°C. This polymer exhibited minimum syneresis (water separation) upon gelation from a solution state when the temperature was raised from room temperature to 37°C, while poly(N-isopropylacrylamide) exhibited considerable syneresis under the same conditions. These properties influence the efficiency of islet entrapment. The copolymer was able to entrap rat islets almost 100%, but the homopolymer entrapped less than 50%. The static insulin secretion of the islets in the copolymer matrix at high glucose concentration (16.5 mM) was comparable to that of control islets, however, the entrapped islets showed prolonged viability in vitro. These results indicate the potential of developing a rechargeable biohybrid pancreas using thermally reversible polymer gels.     

Controlling sol-gel properties enhancing entrapped membrane protein activity through doping additives

Chlorophyllase, a membrane protein, was entrapped in tetramethoxysilane (TMOS)—derived sol-gel and activity examined through a modification of sol-gel properties with doping additives. Polyvinyl alcohol or polyethyleneglycol as uncharged polymeric additives reduced the activity of entrapped chlorophyllase by 60 and 70% respectively, explained by restricted accessibility of chlorophyllase, as revealed by nitrogen desorption experiments. Entrapped chlorophyllase demonstrated 10% activity yield in polyethylenimine-doped gel relative to a polyethylenimine-free control, explained by electrostatic interaction between positively charged polyethylenimine and negatively charged chlorophyllase, considering that polyethylenimine led to a gel with a slightly reduced specific surface area and pore volume, but 20% larger pore size. When chlorophyllase was introduced into sol-gel together with various amounts of glycerol, it demonstrated only slightly lower activity in those gels, even though sol-gel demonstrated decreased specific surface area and pore volume with increasing amount of glycerol. Lipase, as additive, reduced activity of entrapped chlorophyllase, explained by its effect on gel formation, and thus gelation time and gel properties, whereas activity of sol-gel entrapped chlorophyllase associated with monogalactosyl diglyceride (MGDG), was increased by 40%. This study provides an in-depth understanding of the change in sol-gel properties as affected by a wide variety of additives, and the consequent affect on the activity of the membrane protein chlorophyllase.     

Characterization of Sol-gel bioencapsulates for ester hydrolysis and synthesis

Candida rugosa lipase was entrapped in silica sol-gel particles prepared by hydrolysis of methyltrimethoxysilane and assayed by p-nitrophenyl palmitate hydrolysis, as a function of pH and temperature, giving pH optima of 7.8 (free enzyme) and 5.0–8.0 (immobilized enzyme). The optimum temperature for the immobilized enzyme (50–55°C) was 19°C higher than for the free enzyme. Thermal, operational, and storage stability were determined with n-butanol and bytyric acid, giving at 45°C a half-life 2.7 times greater for the immobilized enzyme; storage time was 21 d at room temperature. For ester synthesis, the optimum temperature was 47°C, and high esterification conversions were obtained under repeated batch cycles (half-life of 138 h).     

Entrapping enzyme in a functionalized nanoporous support

The enzyme organophosphorus hydrolase (OPH) was spontaneously entrapped in carboxylethyl- or aminopropyl-functionalized mesoporous silica with rigid, uniform open-pore geometry (30 nm). This approach yielded larger amounts of protein loading and much higher specific activity of the enzyme when compared to the unfunctionalized mesoporous silica and normal porous silica with the same pore size. When OPH was incubated with the functionalized mesoporous silica, protein molecules were sequestered in or excluded from the porous material, depending on electrostatic interaction with the charged functional groups. OPH entrapped in the organically functionalized nanopores showed an exceptional high immobilization efficiency of more than 200% and enhanced stability far exceeding that of the free enzyme in solution. The combination of high protein loading, high immobilization efficiency and stability is attributed to the large and uniform pore structure, and to the optimum environment introduced by the functional groups.     

Phycobiliproteins encapsulated in sol-gel glass

Light transducing phycobiliproteins are encapsulated in optically transparent sol-gel matrices. Absorption and fluorescence spectroscopies are used to characterize the effect of the sol-gel encapsulation on the conformation and aggregation states of the three major phycobiliproteins present in phycobilisomes: phycoerythrin, phycocyanin, and allophycocyanin. It is found that the effects of sol-gel entrapment on the spectroscopic properties are significantly different for the three phycobiliproteins. The results indicate that phycoerythrin undergoes only minor change in its native structure when entrapped in sol-gel. However, significant changes in conformation and aggregation state occur when phycocyanin and allophycocyanin are entrapped in sol-gel matrices. A thin film of sol-gel encapsulated phycoerythrin is also coated on an optical fiber surface and strong fluorescence from the evanescent wave excitation is detected. The potential applications of sol-gel encapsulated phycobiliproteins in biosensors are discussed.     

Silicate Xerogels with Dopant-Induced Chirality

A series of silicate xerogels with entrapped chiral amino acids have been obtained via sol-gel technology. The transparent, glassy samples obtained exhibit chirality in the bulk due to the presence of the entrapped asymmetric molecules. Measurements of the optical activity of the doped xerogel samples revealed that the entrapment did not significantly influence the optical activity observed for liquid solutions of the amino acids. Thus, the sol-gel method enables the preparation of amorphous optical materials exhibiting properties of strictly spatially defined molecular systems. Apart from the obvious optical applications, such porous materials with asymmetric centers might find interesting applications in chiral chemical syntheses and separations.     

Catalytic and spectroscopic properties of cytochrome-c, horseradish peroxidase, and ascorbate oxidase embedded in a sol-gel silica matrix as a function of gelation time

In this study, we investigated the optical features of the redox metal-dependent proteins cytochrome-c, horseradish peroxidase (HRP), and ascorbate oxidase embedded in a sol-gel-processed silica matrix as a function of gelation time. Circular dichroism, absorbance, and fluorescence spectroscopies revealed that the sol-gel process affects the complex structure of the dimeric ascorbate oxidase (although the prosthetic coppers still remain bound to the enzyme) but not that of monomeric cytochrome-c and HRP. Any modifications in ascorbate oxidase occurred in the initial gelation phase; the drying process induced no further alterations and the enzyme remained stable for months. Unfolding-refolding experiments on cytochrome-c revealed severely restricted motility in the protein moiety in the xerogel, the concentrated matrix that forms after drying. The diffusion time of the solvent within the matrix, which regulated the enzyme-substrate reaction rate, depended on the thickness of the monolith, not on the dryness of the specimen.     

Structural Stability of Azurin Encapsulated in Sol-Gel Glasses: A Fluorometric Study

In this study we investigated the structural features of azurin, a blue copper-containing enzyme, upon encapsulation in tetramethoxysilane derived sol-gel glasses. Fluorescence spectroscopy revealed that gelation of inorganic networks does not affect the protein tertiary structure and only after two months solvent phase loss altered protein stability. In case of organically modified sol-gel matrices, the protein stability was reduced after encapsulation into hosts modified by adding 3-Mercaptopropyl-trimethoxysilane, 3-Glycidyloxypropyl-trimethoxysilane and Trimethoxy octylsilane, while it was found to be enhanced in networks doped with 3-Trimethoxysilyl-propyl methacrylate and 3-Aminopropyl-trimethoxysilane. In order to better investigate the effects of silica glasses on azurin stability, unfolding experiments of the protein, in solution or entrapped, were also performed in the presence of both methanol and guanidinium hydrochloride (GdHCl). Our results suggest that the matrix protects azurin against the aggregation induced by alcohol, and that the free energy change value upon unfolding by GdHCl was lower than the value calculated for azurin in solution and was dependent on the surface chemistry of silica matrix.     

Immobilization of Rhodococcus AJ270 and Useof Entrapped Biocatalyst for the Productionof Acrylic Acid

Summary.  Rhodococcus AJ270 is adsorbed by Dowex 1 at 15.4  mg dry weight per g resin with maximum amidase specific activity observed at lower loadings. Bacteria form a monolayer on the resin surface, and adsorption is complete within 2 min. AJ270 can be entrapped in agar and agarose gels (optimum loading: 20 mg dry weight bacteria per cm³ gel). Adsorption and entrapment improve amidase thermal stability 3–4 fold, and entrapment shifts the pH optimum from 8 to 7. Adsorbed and free bacteria show similar values for K _m and V _max, but entrapped bacteria have higher K _m values. Compared with bacteria adsorbed to Dowex, the activity per cm³ of matrix of agar-entrapped AJ270 is eight-fold higher. In stirred-tank reactors, exposure to acrylic acid reduces the amidase activity of the biocatalyst in the hydrolysis of acrylamide. In column reactors, entrapped AJ270 suffers little reduction in amidase activity against 0.25 M acrylamide over 22 h continuous operation. Received November 18, 1999. Accepted December 14, 1999     

Structure and dynamics of lysozyme encapsulated in a silica sol-gel matrix

Proteins entrapped in sol-gel matrices have been extensively studied during the last 15 years, showing that most of them can be encapsulated with retention of their native structure and functionality and with enhanced stability. However, relatively little is known about the structural and dynamical details of the biomolecule-matrix interactions. To achieve this goal, the model protein hen egg white lysozyme (HEWL) has been entrapped in sol-gel matrices prepared from tetraethyl orthosilicate through an alcohol-free sol-gel route, and the photophysical properties of its fluorescent tryptophans have been determined using both steady-state and time-resolved fluorescence techniques. By combining fluorescence spectra, quenching experiments, lifetimes, and time-resolved fluorescence anisotropy measurements, we have obtained information on the structure, dynamics, and solvation properties of the entrapped protein. Our results show that the environment of HEWL within the silica pore as well as its internal dynamics is similar to that in aqueous solution, except that the protein showed no or, depending on conditions, very much slower global motion but retained its internal angularly restricted (hindered) segmental rotation upon entrapment. The experiments carried out at different experimental conditions indicate that, below the isoelectric point of the protein, a strong electrostatic interaction is established between the protein molecule and the negatively charged sol-gel walls, which is ultimately responsible for the total arrest of the overall rotation of the protein, but without significant effect upon its segmental rotational relaxation. The electrostatic nature of the interaction is clearly established since either reducing the positive charge of the protein (by increasing the pH toward its isoelectric point) or increasing the ionic strength of the solution (shielding against the attractive interaction) leads to a situation in which the protein freely rotates within the matrix pore, albeit an order of magnitude more slowly than that in free solution under similar macroscopic solution conditions, and still retains its segmental rotational properties.