Optimal Immobilization Conditions Research Articles

Immobilization of Laccase by Alginate–Chitosan Microcapsules and its Use in Dye Decolorization

Laccase is a ligninolytic enzyme that is widespread in white-rot fungi. Alginate–chitosan microcapsules prepared by an emulsification–internal gelation technique were used to immobilize laccase. Parameters of the immobilization process were optimized. Under the optimal immobilization conditions (2% sodium alginate, 2% CaCl2, 0.3% chitosan and 1:8 ratio by volume of enzyme to alginate), the loading efficiency and immobilized yield of immobilized laccase were 88.12% and 46.93%, respectively. Laccase stability was increased after immobilization. Both the free and immobilized laccase alone showed a very low decolorization efficiency when Alizarin Red was selected for dye decolorization test. When 0.1 mM 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was added into the decolorization system, the decolorization efficiency increased significantly. Immobilized laccase retained 35.73% activity after three reaction cycles. The result demonstrated that immobilized laccase has potential application in dyestuff treatment.

Improved stabilization of microencapsulated Cathepsin B in harsh conditions

A cysteine protease Cathepsin B purified from goat brain was microencapsulated within the CM-cellulose-Ca-alginate membrane having a liquid core in it. The activity yield was maximum under the optimum conditions of immobilization mainly at 4% CM-cellulose, 1% CaCl 2 and 3% alginate concentrations. The encapsulated form of enzyme could retain ∼60% of its original activity during its reusability for more than five successive batch reactions. The physico-chemical characteristics for free and immobilized Cathepsin B were also compared. The optimum pH and temperature of free and immobilized enzyme were 6.0 and 50 °C, and 6.0–6.5 and 55 °C, respectively. The K ′ m value (1.52 mM) for the immobilized enzyme was one and half-fold higher than for the soluble enzyme. The pH and thermal stability as well as organic solvent stability were improved significantly after microencapsulation may make it useful for catalyzing some organic reactions like trans-esterification and trans-amidation.

Preparation and characterization of dendritic composite magnetic particles as a novel enzyme immobilization carrier

Three kinds of composite carriers (S-type, M-type, MS-type) were prepared and assessed their application in immobilizing lipase. The S-type carrier was prepared by copolymerization of tetraethyl orthosilicate and a dendritic monomer derived from 3-aminopropyltriethoxysilane and methyl acrylate, and the MS-type carrier by copolymerization of tetraethyl orthosilicate and 3-aminopropyltriethoxysilane or a dendritic monomer on the surface of magnetic microparticle (Fe 3O 4). The carriers were characterized by means of IR spectrum, titration and particle size analyzer. Subsequent immobilization experiments clearly indicated that the MS-type was the best in lipase immobilization among the three types of carriers. Moreover, the immobilized PPL by simple adsorption could be reused without detectable decline for its activity until up to four cycles, while the immobilized PPL by cross-linking could be reused up to five cycles without any loss in activity. Interestingly, hydrolysis of 2-phenyl-1-propyl acetate catalyzed by PPL immobilized on the S-type and MS-type carriers showed a higher enantioselectivity (18–32% up) than that by free PPL. The optimal immobilization conditions of PPL on MS-type carriers were as follows: 50 mg PPL/g; the stirring time: 60 min; pH 7.0; room temperature.

Development and characterization of an immobilized enzyme reactor (IMER) based on human glyceraldehyde-3-phosphate dehydrogenase for on-line enzymatic studies

Immobilized enzyme reactors (IMERs) for on-line enzymatic studies are useful tool to select specific inhibitors and may be used for direct determination of drug–receptor binding interactions and for the rapid on-line screening to identify specific inhibitors. This technique has been shown to increase the stability of enzymes. The enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an important role in the life cycle of the Trypanosoma cruzi and it has become a key target in the drug discovery program for Chagas’ disease. Crystallographic studies have indicated that there are significant inter-species differences in GAPDH activity and sensitivity. For example the active sites of GAPDH in T. cruzi and humans differ by a substitution of ASP 210 ( T. cruzi) by Leu 194 in human. Based on this information we initiated the study to develop optimal conditions for the covalent immobilization of the human GAPDH enzyme on a modified capillary support (400 mm × 0.10 mm). The chromatographic separation of NAD from NADH was achieved using a RP-Spherex ®-diol-OH (10 cm × 0.46 cm, 10 μm, 100 Å) column. By using multidimensional HPLC chromatography system it was possible to investigate the activity and kinetic parameters of the GAPDH-IMER. The values obtained for D-GA3P and NAD were K m = 3.5 ± 0.2 mM and 0.75 ± 0.04 mM, respectively, and were compared with values obtained with the free enzyme. The activity of the immobilized GAPDH has been preserved for over 120 days.

Immobilization of Papain on the Mesoporous Molecular Sieve MCM-48

The immobilization of papain on the mesoporous molecular sieve MCM-48 (with a pore size of 6.2 nm in diameter) with the aid of glutaraldehyde, and the characteristics of this immobilized papain are described. The optimum conditions for immobilization were as follows: 20 mg native free enzyme/g of the MCM-48 and 0.75 % glutaraldehyde, 2 h at 10–20 °C and pH 7.0. Under these optimum conditions for immobilization, the activity yield [%] of the immobilized enzyme was around 70 %. The influence of the pH on the activity of the immobilized enzyme was much lower compared to the free enzyme. The thermostability of the immobilized enzyme, whose half-life was more than 2500 min, was greatly improved and was found to be significantly higher than that of the free enzyme (about 80 min). The immobilized enzyme also showed good operational stability, and the activity of the immobilized enzyme continued to maintain 76.5 % of the initial activity even after a 12-day continuous operation. Moreover, the immobilized enzyme still exhibited good storage stability. From these results, papain immobilized on the MCM-48 with the aid of glutaraldehyde, can be used as a high-performance biocatalyst in biotechnological processing, in particular in industrial and medical applications.

Comparative Study of Amidase Production by Free and Immobilized <I>Escherichia coli</I> Cells

Escherichia coli NCIM 2569 was evaluated for its potential for amidase production under submerged fermentation. Among the various amide compounds screened, maximum substrate specificity and enzyme yield (8.1 U/mL) were obtained by using 1% acetamide. Fermentation was carried out at 30 degrees C in shake-flask culture under optimized process conditions. A maximum of 0.52 U/mL of intracellular amidase activity was also obtained from cells incubated for 24 h. Studies were also performed to elucidate the optimal conditions (gel concentration, initial biomass, curing period of beads, and calcium ion concentration in the production medium) for immobilization of whole cells. By using E. coli cells entrapped in alginate, a maximum of 6.2 U/mL of enzyme activity was obtained after 12 h of incubation under optimized conditions. Using the immobilized cells, three repeated batches were carried out successfully, and 85% of the initial enzyme activity was retained in the second and third batches. The study indicated that the immobilized E. coli cells offered certain advantages such as less time for maximum enzyme production, more stability in the enzyme production rate, and repeated use of the biocatalyst.

Synthesis of diagnostic test kits for syphilis with polymeric particles

This investigation aimed to derive alternative guidelines for syphilis. In the first step of this study monodisperse PGMA particles were synthesised by dispersion polymerization of the GMA monomer by using AIBN as initiator and PAA as stabilizator. A series of characterization experiments were done. In the second step of the study the characterized particles were used as microcarriers for the immobilization of the natural antigen for the purpose of diagnosing syphilis. The covalent binding method was used and GA was utilized as a spacer arm. The optimum immobilization conditions for the binding of natural antigen onto the particles were determined. In the third and the last step of the study the agglutination tests were realized with in vitro experiments by using both serum samples taken from syphilis patients and positive controls. After these experiments the kits chosen among the ones showing high performance were compared with the commercial RPR kit being used widely by reacting with serially diluted positive controls and patient serums. After this comparison the performances of the synthesized kits were found as satisfactory as the commercial kits.

Continuous production of monoacylglycerols by glycerolysis of palm olein with immobilized lipase

Nine commercial lipases from Pseudomonas sp. (lipase PS), Pseudomonas fluorescens (lipase AK), Candida rugosa (lipase AY), Rhizopus delemar (lipase D), Mucor javanicus (lipase M), Rhizopus oryzae (lipase F), C. rugosa (lipase OF) Alcaligenes sp. (lipase PL) and Chromobacterium viscosum (lipase LP) were screened for production of monoacylglycerols (MAG). Lipase PS was the most suitable enzyme for glycerolysis of palm olein with glycerol. This lipase had hydrolytic activity 10.42 U/mg and provided a high yield of MAG with 28.05% at 45 °C. Celite, silica gel, CaCO 3, Accurel EP100 and activated charcoal were used as supports to immobilize lipase PS. Accurel EP100 (<200 μm) was the best support. The optimum conditions for immobilization included the enzyme concentration of 50 U/ml and immobilization temperature at 30 °C for 30 min. When 5.0 ml enzyme solution was mixed with 0.5 g support the immobilized activity was 0.23 U/mg support and immobilized yield was 45.38%. The immobilized lipase PS (IM-PS) on Accurel had optimal activity at 45–65 °C and more than 90.0% of the activity remained after incubated at 45 °C for 24 h. In batch production 20.74% MAG was obtained at 45 °C for 24 h. The continuous production of MAG was performed with IM-PS (350 U) in the packed-bed reactor (PBR) (0.68 cm ID, 25 cm long) and a continuous stirred-tank reactor (CSTR) (4.5 cm ID, 6.0 cm height) for 96 h at 45 °C. When the flow rate of the substrate mixture was 0.02 ml/min the average yields of MAG were 14.01 and 14.34% in PBR and CSTR, respectively.

Penicillium sp. ZD-Z1 chitosanase immobilized on DEAE cellulose by cross-linking reaction

Chitosanase obtained fromPenicillium sp.ZD-Z1 was immobilized on DEAE cellulose with glutaraldehyde by cross-linking reaction. The optimal conditions of immobilization were as follows: 0.1 g DEAE cellulose was treated with 5 ml 5% glutaraldehyde solution; then 2.3 mg chitosanase was immobilized on the carrier. The optimal temperature and pH was 60 °C and 4.0, and the K m value was 18.87 g/L. Under optimal conditions, the activity of immobilized enzyme is 1.5 U/g, and the recovery of enzyme activity is 81.3%. After immobilization, the optimal temperature and K m value increased (from 50 °C to 60 °C, from 2.49 g/L to 18.87 g/L), whereas the optimal pH was reduced (from 5.0 to 4.0). The enzyme activity loss was less than 20% after 10 times batch reaction; the immobilized enzyme showed good operation stability.

Covalent immobilization of oligonucleotides on electrodes

32P-labeled DNA oligomers were immobilized onto self-assembled monolayers formed by carboxy-modified alkylthiols on gold electrodes. The optimal conditions were evaluated by comparing of surface density of oligonucleotides measured for different immobilization conditions before and after desorption of physically adsorbed nucleotides. The results demonstrate that the physical adsorption depends strongly on immobilization conditions being typically 10 times higher than the chemical immobilization. Under optimal immobilization conditions, the surface density of oligonucleotides was more than 20% of the value calculated for a densely packed monolayer.

A new approach for immobilization of oligonucleotides onto piezoelectric quartz crystal for preparation of a nucleic acid sensor for following hybridization

The aim of this study is to develop a nucleic acid sensor based on piezoelectric crystal microbalance system (QCM) for following hybridization. Piezoelectric quartz crystal surfaces were first treated in a glow–discharge apparatus with ethylene diamine (EDA) plasma at 15 W (discharge power), 2.5 min (incubation time) and 35 ml/min (monomer flow rate) to create amino groups on the crystal surfaces. The thickness of the EDA–plasma film formed was about 43±24 Å. Then, the amino groups on the crystal surfaces were converted to aldehyde groups by reacting the amino groups with glutaraldehyde (GA) at different conditions. A GA concentration of 2.5% and an incubation time of 2 h were selected as optimal values at this step, corresponding to a GA surface concentration of about 270 ng/cm 2. A double strand Oligonucleotides, having one extra base on 5′-end of one of the complementary strands, were immobilized through the amino groups of this base onto the GA-modified crystals. Optimal immobilization conditions were as follows: oligonucleotide concentration: 1 μg/ml; time: 3 h; pH: 9.2 carbonate buffer; ionic strength: 0.1; and temperature: 20 °C. The QCM sensor carrying the covalently bound strand was used in the hybridization experiments, which showed that equilibrium is achieved in about 5 min, and the frequency shift measured is related to the concentration of the target strand to be measured within the medium.

Immobilization of fish chromatophores for use as a micro-biosensor for biological toxins

Chromatophores isolated from the Siamese fighting fish, Betta splendens represent a class of living cells that provide a vivid color response to microbial pathogens and environmental toxins. The selection of the most appropriate microcarrier and the development of the optimal technique for the chromatophore immobilization in order to enable directed transport of the sensor cells throughout microchannels of the biosensor, as well to preserve the cell survival and its functionality was studied. Microcarriers derived from glass, polystyrene and gelatin (collagen) were tested as substrates for chromatophore attachement. Gelatin microcarriers were found to be the most suitable, due to high attachment efficiency (95% of attached cells), preservation of the cell viability and enhanced cell sensitivity. The optimum conditions for fish cell immobilization on collagen microcarriers were determined based on the cell-to-microcarrier bead ratio and the pH of the solution. The rate of cell attachment to the gelatin microcarrier followed first-order kinetics. Pretreatment of the gelatin beads with fibronectin, known as a cell attachment-promoting agent, resulted in a 10% higher attachment rate constant (k).

Studies on Penicillium sp. ZDZ1 chitosanase immobilized on chitin by cross-linking reaction

Chitosanase was immobilized on chitin with glutaraldehyde by cross-linking reaction and the immobilization conditions and characterization of the immobilized enzyme were carried out. The optimal conditions for immobilization were as follows: 0.1 g chitin was treated with 5 ml 5% solution of glutaraldehyde and 2 mg chitosanase was immobilized on the carrier. Optimal temperature and pH for the immobilized enzyme were 60° C and 4.0, respectively. Kinetic parameter K m was 17.66 g/l. The immobilized enzyme showed better temperature stability than the free enzyme and its operation and storage stability were good.

Glow-discharge treated piezoelectric quartz crystals as immunosensors for HSA detection

The aim of this study is to develop an immunoaffinity sensor based on piezoelectric crystals for human serum albumin (HSA) detection in aqueous media. Quartz crystals were treated with ethylene diamine (EDA) plasma in a glow-discharge apparatus in order to substitute amino groups on their surfaces. Then anti-HSA antibodies were immobilized via these amino groups by using glutaraldehyde (GA) as cross-linker. Immobilization of the antibody on the quartz crystal was examined for different pH, antibody concentration and treatment time. The optimum conditions for anti-HSA immobilization were evaluated by the measurements of the activity of the surface against HSA. The optimum values of pH, antibody concentration and treatment time were found 6.2, 0.15 mg/ml and 2 h, respectively. For detection of HSA into the solution, two methods were used. In the first (dip and dry) method, the frequency shifts were measured in air after the 1 h interaction of the anti-HSA immobilized crystals with HSA solution. In the other (direct) method, the frequency shifts were followed continuously for 60 min. while the probe was immersed in the HSA solution. An increase for the frequency shifts was observed with increasing of HSA concentration of 16–128 μg/ml. Both the immobilization and antibody-interaction conditions were found important on the extend of these specific interaction. The relations between the HSA concentrations and frequency shifts were exponential in both methods.

Alginate-immobilized lipase by electrostatic extrusion for the purpose of palm oil hydrolysis in lecithin/isooctane system

Lipase from Candida rugosa was immobilized in alginate beads for possible application in non-aqueous or microaqueous reaction systems. An electrostatic droplet generation technique was used for production of small diameter (<1 mm) lipase-alginate beads. This technique provided negligible loss of the lipase (immobilization efficiencies were 98.2–99.2%). Under optimal immobilization conditions (applied potential 4.9 kV, needle gauge 21, 2% sodium alginate solution) the lipase-alginate beads, 0.65 mm in diameter, retained enzyme activity equivalent to 75% that of free lipase. The activity of the immobilized lipase was verified in the reaction of palm oil hydrolysis in a lecithin/isooctane system. The reaction rate with alginate-immobilized lipase was lower than with the free enzyme but the final conversions were approximately the same (∼74%). Immobilized lipase could be used for up to three reaction cycles with little loss of activity.

Immobilization of invertase onto crosslinked poly(p‐chloromethylstyrene) beads

AbstractInvertase was immobilized onto poly(p‐chloromethylstyrene) (PCMS) beads that were produced by a suspension polymerization with an average size of 186 μm. The beads had a nonporous but reasonably rough surface. Because of this, a reasonably large external surface area (i.e., 14.1 m2/g) could be achieved with the proposed carrier. A two‐step functionalization protocol was followed for the covalent attachment of invertase onto the bead surface. For this purpose, a polymeric ligand that carried amine groups, polyethylenimine (PEI), was covalently attached onto the bead surface by a direct chemical reaction. Next, the free amine groups of PEI were activated by glutaraldehyde. Invertase was covalently attached onto the bead surface via the direct chemical reaction between aldehyde and amine groups. The appropriate enzyme binding conditions and the batch‐reactor performance of the immobilized enzyme system were investigated. Under optimum immobilization conditions, 19 mg of invertase was immobilized onto each gram of beads with 80% retained activity after immobilization. The effects of pH and temperature on the immobilized invertase activity were determined and compared with the free enzyme. The kinetic parameters KM and VM were determined with the Michealis–Menten model. KM of immobilized invertase was 1.75 folds higher than that of the free invertase. The immobilization caused a significant improvement in the thermal stability of invertase, especially in the range of 55–65°C. No significant internal diffusion limitation was detected in the immobilized enzyme system, probably due to the surface morphology of the selected carrier. This result was confirmed by the determination of the activation energies of both free and immobilized invertases. The activity half‐life of the immobilized invertase was approximately 5 times longer than that of the free enzyme. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 83: 1268–1279, 2002

Immobilization of urease in conducting thiophene-capped poly(methyl methacrylate)/pyrrole matrices

Urease was immobilized in conducting polypyrrole and block copolymers of thiophene-capped poly(methyl methacrylate) matrices by electropolymerization. Immobilization of the enzyme was achieved by application of 1.0 V constant potential in a solution of 0.02 M pyrrole, 2 mg/ml urease, 0.5 mg/ml supporting electrolyte. The optimum immobilization conditions, electrolysis time, electrolysis medium, enzyme concentration during electrolysis, type of supporting electrolyte were determined. The kinetic behaviors of free and immobilized urease were determined.

Biodegradation of quinoline by gel immobilized Burkholderia sp.

Burkholderia sp, a Gram-negative, rod-shaped, aerobe, capable of degrading quinoline was immobilized in calcium alginate gel beads and used for degradation of quinoline in aqueous solution in the reactor. The optimal conditions for immobilization of the microorganism, such as alginate concentration, calcium ion concentration, initial cell loading, hardening time and bead size, were determined with a view to improving the quinoline degradation rate. The characteristics of quinoline degradation by immobilized microbial cells were investigated. The repeated use of immobilized cells for quinoline degradation was performed and the results revealed that the bioactivity of immobilized cells was stable over 100 h in the repeated batch cultivation for quinoline degradation.

Studies of poly(vinyl acetate-co-diviyl benzene) beads as a carrier for the immobilization of penicillin acylase and the kinetics of immobilized penicillin acylase

Poly(vinyl acetate-co-diviyl benzene) beads were synthesized via a novel polymerization method. The amount of hydroxide radical could reach 11.5 mmol g −1 (wet) after the beads were catalyzed by NaOH in methanol at 18–20°C for 24 h. The activators such as SESA, glutaraldehyde and p-benzoquinone were used to immobilize penicillin acylase. The penicillin acylase immobilized with SESA exhibited better stability than that with other activators. The successively immobilization could increase the activity of immobilized penicillin acylase about two times and the activity yield 10%, respectively. At the optimal conditions of immobilization, the activity of immobilized penicillin acylase amounted to 812–1200 unit per gram (wet). The kinetics of immobilized penicillin acylase was also studied. The reaction constants, K m , K iPAA , K iAPA and K s for immobilized penicillin acylase were equal to 0.033, 0.013, 0.009 and 0.081 mmol/ml, respectively.

Cell immobilization of Trichosporon cutaneum strain with phenol degradation ability on new modified polymer carriers

Chemical immobilization of Trichosporon cutaneum R57 cells onto modified polyacrylonitrile granules and fibres was carried out. The polymer carriers were modified in water solutions of NaOH and 1,2-diaminoethane. The concentration of amino groups obtained by the modification was determined. The optimal conditions for chemical immobilization of cells of T. cutaneum R57 with glutaraldehyde were found to be: 40% NaOH concentration, 46.8 mg/ml initial cell suspension, 24 h immobilization time and pH 6. The change in protein concentration of free and immobilized cells was studied during a full biodegradation cycle. The rate of phenol biodegradation by T. cutaneum R57 cells immobilized on porous polyacrylonitrile (PAN) fibres was higher than that obtained using modified PAN beds.