Showing papers on "Immobilized enzyme published in 2007"
TL;DR: In all cases, enzyme engineering via immobilization techniques is perfectly compatible with other chemical or biological approaches to improve enzyme functions and the final success depend on the availability of a wide battery of immobilization protocols.
3,016 citations
TL;DR: Different methods for the immobilization of enzymes are critically reviewed, with emphasis on relatively recent developments, such as the use of novel supports, e.g., mesoporous silicas, hydrogels, and smart polymers, novel entrapment methods and cross-linked enzyme aggregates (CLEAs).
Abstract: Immobilization is often the key to optimizing the operational performance of an enzyme in industrial processes, particularly for use in non-aqueous media. Different methods for the immobilization of enzymes are critically reviewed. The methods are divided into three main categories, viz. (i) binding to a prefabricated support (carrier), (ii) entrapment in organic or inorganic polymer matrices, and (iii) cross-linking of enzyme molecules. Emphasis is placed on relatively recent developments, such as the use of novel supports, e.g., mesoporous silicas, hydrogels, and smart polymers, novel entrapment methods and cross-linked enzyme aggregates (CLEAs).
1,857 citations
TL;DR: A novel, versatile and effective methodology for enzyme immobilization as CLEAs (cross-linked enzyme aggregates) is described, which is exquisitely simple and amenable to rapid optimization.
Abstract: The key to obtaining an optimum performance of an enzyme is often a question of devising an effective method for its immobilization. In the present review, we describe a novel, versatile and effective methodology for enzyme immobilization as CLEAs (cross-linked enzyme aggregates). The method is exquisitely simple (involving precipitation of the enzyme from aqueous buffer followed by cross-linking of the resulting physical aggregates of enzyme molecules) and amenable to rapid optimization. We have shown it to be applicable to a wide variety of enzymes, including, in addition to a wide variety of hydrolases, lyases, e.g. nitrile hydratases and oxynitrilases, and oxidoreductases such as laccase and galactose oxidase. CLEAs are stable, recyclable catalysts exhibiting high catalyst productivities. Because the methodology is essentially a combination of purification and immobilization into one step, the enzyme does not need to be of high purity. The technique is also applicable to the preparation of combi-CLEAs, containing two or more enzymes, for use in one-pot, multistep syntheses, e.g. an oxynitrilase/nitrilase combi-CLEA for the one-pot conversion of benzaldehyde into (S)-mandelic acid, in high yield and enantiomeric purity.
391 citations
TL;DR: Different methods for the immobilization of enzymes are critically reviewed, with emphasis on relatively recent developments, such as the use of novel supports, e.g., mesoporous silicas, hydrogels, and smart polymers, novel entrapment methods and cross-linked enzyme aggregates (CLEAs).
Abstract: Immobilization is often the key to optimizing the operational performance of an enzyme in industrial processes, particularly for use in non-aqueous media. Different methods for the immobilization of enzymes are critically reviewed. The methods are divided into three main categories, viz. (i) binding to a prefabricated support (carrier), (ii) entrapment in organic or inorganic polymer matrices, and (iii) cross-linking of enzyme molecules. Emphasis is placed on relatively recent developments, such as the use of novel supports, e.g., mesoporous silicas, hydrogels, and smart polymers, novel entrapment methods and cross-linked enzyme aggregates (CLEAs).
273 citations
TL;DR: “tailor-made” heterofunctional epoxy supports are designed and the performance of the immobilization protocols are described here.
Abstract: Immobilization of enzymes and proteins on activated supports permits the simplification of the reactor design and may be used to improve some enzyme properties. In this sense, supports containing epoxy groups seem to be useful to generate very intense multipoint covalent attachment with different nucleophiles placed on the surface of enzyme molecules (e.g., amino, thiol, hydroxyl groups). However, the intermolecular reaction between epoxy groups and soluble enzymes is extremely slow. To solve this problem, we have designed "tailor-made" heterofunctional epoxy supports. Using these, immobilization of enzymes is performed via a two-step process: (i) an initial physical or chemical intermolecular interaction of the enzyme surface with the new functional groups introduced on the support surface and (ii) a subsequent intense intramolecular multipoint covalent reaction between the nucleophiles of the already immobilized enzyme and the epoxy groups of the supports. The first immobilization may involve different enzyme regions, which will be further rigidified by multipoint covalent attachment. The design of some heterofunctional epoxy supports and the performance of the immobilization protocols are described here. The whole protocol to have an immobilized and stabilized enzyme could take from 3 days to 1 week.
257 citations
TL;DR: In this article, a chitosan nanofibrous membrane with stabilized morphology was used for enzyme immobilization and the results showed that the observed lipase loading on this membrane was up to 63.6 mg/g and activity retention of the immobilized lipase was 49.8% under the optimum condition.
238 citations
TL;DR: The glucose biosensor shows fast amperometric response (3s) with the sensitivity of 446.2nA/mM, detection limit of 24 microM and wide concentration range of 30 microM to 5mM and exhibits good stability, reproducibility and long life time.
225 citations
TL;DR: It was observed that the number of lysine residues that took part in the immobilization process was a consequence of the type of support and reaction time of the experimental conditions, and that the increasing of the thermal stability of the derivatives was correlated with a increasing number ofLysines residues involved in a multipoint covalent attachment.
203 citations
TL;DR: The possibilities of a directed immobilization of mutated enzymes (change of an amino acid by cysteine on specific points of the protein surface) on tailor-made disulfide-epoxy supports will be discussed as an almost-ideal procedure to achieve very intense and very efficient rigidification of a desired region of industrial enzymes.
Abstract: Multipoint covalent immobilization of enzymes (through very short spacer arms) on support surfaces promotes a very interesting 'rigidification' of protein molecules. In this case, the relative positions of each residue of the enzyme involved in the immobilization process have to be preserved unchanged during any conformational change induced on the immobilized enzyme by any distorting agent (heat, organic solvents etc.). In this way, multipoint covalent immobilization should induce a very strong stabilization of immobilized enzymes. Epoxy-activated supports are able to chemically react with all nucleophile groups placed on the protein surface: lysine, histidine, cysteine, tyrosine etc. Besides, epoxy groups are very stable. This allows the performance of very long enzyme-support reactions, enabling us to get very intense multipoint covalent attachment. In this way, these epoxy supports seem to be very suitable to stabilize industrial enzymes by multipoint covalent attachment. However, epoxy groups exhibit a low intermolecular reactivity towards nucleophiles and hence the enzymes are not able to directly react with the epoxy supports. Thus a rapid physical adsorption of enzymes on the supports becomes a first step, followed by an additional rapid 'intramolecular' reaction between the already adsorbed enzyme and the activated support. In this situation, a suitable first orientation of the enzyme on the support (e.g. through regions that are very rich in nucleophiles) is obviously necessary to get a very intense additional multipoint covalent immobilization. The preparation of different 'generations' of epoxy supports and the design of different protocols to fully control the first interaction between enzymes and epoxy supports will be reviewed in this paper. Finally, the possibilities of a directed immobilization of mutated enzymes (change of an amino acid by cysteine on specific points of the protein surface) on tailor-made disulfide-epoxy supports will be discussed as an almost-ideal procedure to achieve very intense and very efficient rigidification of a desired region of industrial enzymes.
201 citations
TL;DR: It is demonstrated that HMMS can be efficiently employed as host materials for enzyme immobilization leading to highly enhanced stability of the immobilized enzymes with high enzyme loading and activity.
Abstract: α-chymotrypsin (CT) and lipase (LP) were immobilized in hierarchically-ordered mesocellular mesoporous silica (HMMS) in a simple but effective way for the enzyme stabilization, which was achieved by the enzyme adsorption followed by glutaraldehyde (GA) crosslinking. This resulted in the formation of nanometer scale crosslinked enzyme aggregates (CLEAs) entrapped in the mesocellular pores of HMMS (37 nm), which did not leach out of HMMS through narrow mesoporous channels (13 nm). CLEA of α-chymotrypsin (CLEA-CT) in HMMS showed a high enzyme loading capacity and significantly increased enzyme stability. No activity decrease of CLEA-CT was observed for 2 weeks under even rigorously shaking condition, while adsorbed CT in HMMS and free CT showed a rapid inactivation due to the enzyme leaching and presumably autolysis, respectively. With the CLEA-CT in HMMS, however, there was no tryptic digestion observed suggesting that the CLEA-CT is not susceptible to autolysis. Moreover, CLEA of lipase (CLEA-LP) in HMMS retained 30% specific activity of free lipase with greatly enhanced stability. This work demonstrates that HMMS can be efficiently employed as host materials for enzyme immobilization leading to highly enhanced stability of the immobilized enzymes with high enzyme loading and activity. Biotechnol. Bioeng. 2007;96: 210–218. © 2006 Wiley Periodicals, Inc.
193 citations
TL;DR: In this article, hydrotalcite and four different types of zeolites were used as metarial metarial agents, and the size and type of the zeolite particles did not affect the amount of protein adsorbed.
TL;DR: This review presents various surface engineerings for PAN-based membranes, which mainly refers to grafting polymerization, partially hydrolysis, macromolecule immobilization, and enzyme immobilization.
TL;DR: The immobilized urease had good storage and operational stability and good reusability, properties that offer potential for practical application.
Abstract: Urease was covalently immobilized on glutaraldehyde-pretreated chitosan membranes. The optimum immobilization conditions were determined with respect to glutaraldehyde pretreatment of membranes and to reaction of glutaraldehyde-pretreated membranes with urease. The immobilized enzyme retained 94% of its original activity. The properties of free and immobilized urease were compared. The Michaelis constant was about five times higher for immobilized urease than for the free enzyme, while the maximum reaction rate was lower for the immobilized enzyme. The stability of urease at low pH values was improved by immobilization; temperature stability was also improved. The optimum temperature was determined to be 65°C for the free urease and 75°C for the immobilized form. The immobilized enzyme had good storage and operational stability and good reusability, properties that offer potential for practical application.
TL;DR: In this article, a laccase from Trametes versicolor was immobilized on magnetic mesoporous silica spheres through physical adsorption and covalent attachment methods.
Abstract: Magnetic mesoporous silica spheres (MSS) with large pore size were prepared by reducing Fe3+-containing mesoporous silica spheres. Laccase from Trametes versicolor was immobilized on magnetic mesoporous silica spheres through physical adsorption and covalent attachment methods. Laccase oxidizes 2,2′-azinobis(3-ethylbenzthiazolin-6-sulfonate) (ABTS) to the green cation radical (ABTS•+) as a model system; the immobilized laccase retained the activity and exhibited higher resistance to pH changes and thermal stability. The immobilized laccase obtained through covalent attachment almost has no leaching and can retain above 70% of activity after 10 consecutive operations. More interesting, the immobilized laccase can be separated quickly using an external magnetic field. Therefore, the magnetite-containing mesoporous silica spheres are a promising support for enzyme immobilization.
TL;DR: The cloning and expression of L-asparaginase from Erwinia chrysanthemi 3937 (ErL-ASNase) in Escherichia coli BL21(DE3)pLysS is reported and it is found that the enzyme can be efficiently immobilized on epoxy-activated Sepharose CL-6B.
TL;DR: This lipase immobilization method shows the best performance among various immobilized lipase systems using the same source of lipase and substrate when considering protein loading, activity retention, and kinetic parameters.
Abstract: Polyacrylonitrile (PAN) nanofibers could be fabricated by electrospinning with fiber diameter in the range of 150–300 nm, providing huge surface area for enzyme immobilization and catalytic reactions. Lipase from Candida rugosa was covalently immobilized onto PAN nanofibers by amidination reaction. Aggregates of enzyme molecules were found on nanofiber surface from field emission scanning electron microscopy and covalent bond formation between enzyme molecule and the nanofiber was confirmed from FTIR measurements. After 5 min activation and 60 min reaction with enzyme-containing solution, the protein loading efficiency was quantitative and the activity retention of the immobilized lipase was 81% that of free enzyme. The mechanical strength of the NFM improved after lipase immobilization where tensile stress at break and Young's modulus were almost doubled. The immobilized lipase retained >95% of its initial activity when stored in buffer at 30 °C for 20 days, whereas free lipase lost 80% of its initial activity. The immobilized lipase still retained 70% of its specific activity after 10 repeated batches of reaction. This lipase immobilization method shows the best performance among various immobilized lipase systems using the same source of lipase and substrate when considering protein loading, activity retention, and kinetic parameters.
TL;DR: The GOD/nano-CaCO3-based sensor exhibited a marked improvement in thermal stability compared to other glucose biosensors based on inorganic host matrixes and the effects of the applied potential as well as common interfering compounds on the amperometric response of the sensor were investigated and discussed herein.
TL;DR: This work describes development and optimization of a generic method for the immobilization of enzymes in chemically synthesized gold polypyrrole (Au-PPy) nanocomposite and their application in amperometric biosensors, which has proved to be an effective way for stable enzyme attachment.
TL;DR: Photopatterning enzymes on polymer monoliths provides a simple technique for preparing spatially localized multiple-enzyme microreactors capable of directional synthesis.
Abstract: A method for photopatterning multiple enzymes on porous polymer monoliths within microfluidic devices has been developed and used to perform spatially separated multienzymatic reactions. To reduce nonspecific adsorption of enzymes on the monolith, its pore surface was modified by grafting poly(ethylene glycol), followed by surface photoactivation and enzyme immobilization in the presence of a nonionic surfactant. Characterization of bound horseradish peroxidase (HRP) was carried out using a reaction in which the steady-state profiles of the fluorescent reaction product could be measured in situ and then analyzed using a plug-flow bioreactor model to determine the observed maximum reaction rate and Michaelis constant. The Michaelis constant of 1.9 μmol/L agrees with previously published values. Mass-transfer limitations were evident at relatively low flow rates but were absent at higher flow rates. Sequential multienzymatic reactions were demonstrated using the patternwise assembly of two- and three-enzyme...
TL;DR: In this paper, gold nanoparticles-mesoporous silica composite (GNPs-MPS) is developed as a novel enzyme immobilization matrix for biosensor construction, and the synthesis process of the composite is monitored by UV-vis spectroscopy and the product is characterized by transmission electron microscopy (TEM) measurement.
Abstract: Gold nanoparticles-mesoporous silica composite (GNPs-MPS) is developed as a novel enzyme immobilization matrix for biosensor construction The mesoporous silica SBA-15 is chosen and the GNPs-SBA-15 is formed from AuCl4− adsorbed H2N-SBA-15 by NaBH4 reduction The synthesis process of the composite is monitored by UV–vis spectroscopy and the product is characterized by transmission electron microscopy (TEM) measurement An amperometric glucose biosensor is built by immobilizing IO4−-oxidized-glucose oxidase (IO4−-oxidized-GOD) on GNPs-MPS modified Au electrode using 2-aminoethanethiol as a cross-linker Cyclic voltammetry (CV) and amperometry are employed to investigate the catalytic behavior of the biosensor to the oxidation of glucose As a result, the biosensor exhibits an excellent bioelectrocatalytic response to glucose with a fast response time less than 7 s, a broad linear range of 002–14 mM, high sensitivity of 61 μA mM−1 cm−2, as well as good long-term stability and reproducibility These performances could be ascribed to the GNPs-MPS's features, such as excellent conductivity, large surface area and good biocompatibility
TL;DR: This experimental work represents a first attempt at construction of a new carbon nanotube-tyrosinase based biosensor able to determine the concentration of BPA, one of the most ubiquitous and hazardous endocrine disruptors which can pollute the drinking and surface water, as well as many products of the food chain.
01 Jan 2007
TL;DR: It was shown that the relative activities of immobilized enzymes are higher than free enzymes over broader pH and temperature ranges, and the Km and Vmax values with immobilization enzymes were larger than those with free enzymes, except for the Vmax value with glucoamylase.
Abstract: Thermal and pH stabilities of free and immobilized α-amylase, β-amylase, and glucoamylase were compared, in which immobilization support was prepared by equal weights of chitosan and activated clay and were cross-linked with glutaraldehyde. It was shown that the relative activities of immobilized enzymes are higher than free enzymes over broader pH and temperature ranges. α-Amylase and glucoamylase immobilized on composite bead maintained 81% of their original activities after 50 times of repeated use. Thermal deactivation energies of free and immobilized enzymes were obtained according to the Arrhenius’ equation. The Michaelis constant (Km) and the maximum rate of starch hydrolysis reaction (Vmax) were also calculated according to the Lineweaver–Burk plot. It was found that the Km and Vmax values with immobilized enzymes were larger than those with free enzymes, except for the Vmax value with glucoamylase.
TL;DR: In this article, α-amylase was covalently immobilized onto phthaloyl chloride-containing amino group functionalized glass beads and was successfully performed under very mild conditions.
TL;DR: This communication describes the use of a molecular hydrogel to immobilize enzymes for catalyzing reactions in an organic solvent to attain superactivity and exceptional stability.
01 Jan 2007
TL;DR: In this paper, a-amylase was covalently immobilized onto phthaloyl chloride-containing amino group functionalized glass beads and was found 25.2 ± 3.1 mg/g glass support.
Abstract: In this study, a-amylase was covalently immobilized onto phthaloyl chloride-containing amino group functionalized glass beads. In this procedure, amide bonds were formed between amino groups on the protein and acid chloride groups on the glass surface. The surface modified beads were characterized using Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), energy dispersion spectrum (EDS) and UV–Vis spectroscopy. Immobilization was successfully performed under very mild conditions (15 C, 4 h). The amount of covalently bound a-amylase was found 25.2 ± 3.1 mg/g glass support. The optimum pH value for the free amylase was at pH 6.5. The optimum pH of the immobilized enzyme was shifted 1.0 pH unit to the acidic region. The immobilized a-amylase exhibited better thermostability than the free one. The free enzyme lost all its activity with in 15 days. Covalently bound amylase was stable up to 5 days and lost only 20% of activity in 25 days. The covalently bound enzyme demonstrated more than 98% activity after 6 runs and 81.4% activity after 25 runs. 2007 Elsevier Ltd. All rights reserved.
TL;DR: Milk clotting activity was used for evaluation of the applicability of pepsin immobilization to industrial process and optimum milk clotting temperature was found as 40 °C for free pepin and 50 ° C for immobilized pepsIn.
TL;DR: In this article, a membrane-less glucose/O 2 biofuel cell based on oxygen contactor was developed, in which both enzymes and mediators were immobilized within electropolymerized polypyrrole polymers.
TL;DR: In this paper, the enzymes α-amylase, glucoamylases and invertase were applied to acid activated montmorillonite (K 10) via adsorption and grafting using glutaraldehyde as spacer.
TL;DR: The ability of the nanoparticle-based sensor to reliably measure concentrations of the organophosphate pesticide carbofuran at nM concentrations was demonstrated by monitoring the inhibition of the hydrolysis of acetylthiocholine.
TL;DR: In this article, α-Amylase was immobilized on zirconia via adsorption, and the support and the immobilized enzymes were characterized using XRD, IR spectra and N2 adaption studies.