Showing papers on "Immobilized enzyme published in 2000"
TL;DR: Immobilization of oxidative enzymes on porous ceramic supports or resins did not adversely affect their stability and showed a good potential for degradation of environment persistent aromatics.
Abstract: A number of oxidative enzymes from bacteria, fungi and plants have been reported to play an important role in numerous waste treatment applications. Peroxidases and/or phenoloxidases can act on specific recalcitrant pollutants by precipitation or transforming to other products and permitting a better final treatment of the waste. Improvement in the useful life and thereby a reduction in treatment cost has been accomplished through enzyme immobilization. Horseradish peroxidase, lignin peroxidase and manganese peroxidase mineralize a variety of recalcitrant aromatic compounds. Immobilization of these enzymes on porous ceramic supports or resins did not adversely affect their stability and showed a good potential for degradation of environment persistent aromatics. Tyrosinase, which catalyzes the hydroxylation of phenols and dehydrogenation of o-diphenols, in an immobilized form exerted an excellent phenol removal. Laccase is capable of eliminating the phenols through polymerization process, however, the presence of mediator such as ABTS and HBT degraded phenol by oxidative process. Many applications with oxidative enzymes and plant materials in effluent as in soil remediation will be discussed.
894 citations
TL;DR: Tametes hirsuta and a purified laccase from this organism were able to degrade triarylmethane, indigoid, azo, and anthraquinonic dyes and reduced their toxicities by up to 80%.
Abstract: Trametes hirsuta and a purified laccase from this organism were able to degrade triarylmethane, indigoid, azo, and anthraquinonic dyes. Initial decolorization velocities depended on the substituents on the phenolic rings of the dyes. Immobilization of the T. hirsuta laccase on alumina enhanced the thermal stabilities of the enzyme and its tolerance against some enzyme inhibitors, such as halides, copper chelators, and dyeing additives. The laccase lost 50% of its activity at 50 mM NaCl while the 50% inhibitory concentration (IC(50)) of the immobilized enzyme was 85 mM. Treatment of dyes with the immobilized laccase reduced their toxicities (based on the oxygen consumption rate of Pseudomonas putida) by up to 80% (anthraquinonic dyes). Textile effluents decolorized with T. hirsuta or the laccase were used for dyeing. Metabolites and/or enzyme protein strongly interacted with the dyeing process indicated by lower staining levels (K/S) values than obtained with a blank using water. However, when the effluents were decolorized with immobilized laccase, they could be used for dyeing and acceptable color differences (DeltaE*) below 1.1 were measured for most dyes.
729 citations
TL;DR: In this paper, an enzyme, horseradish peroxidase (HRP), was adsorbed in the manner of the single immersion method on the silica mesoporous materials FSM-16, MCM-41, and SBA-15 with various pore diameters from 27 to 92 A, and their enzymatic activities in an organic solvent and thermal stabilities were studied.
Abstract: An enzyme, horseradish peroxidase (HRP), was adsorbed in the manner of the single immersion method on the silica mesoporous materials FSM-16, MCM-41, and SBA-15 with various pore diameters from 27 to 92 A, and their enzymatic activities in an organic solvent and the thermal stabilities were studied. FSM-16 and MCM-41 showed a larger amount of adsorption of HRP than SBA-15 or silica gel when the pore sizes were larger than the 50 A. The increased enzyme adsorption capacity may be due to the surface characteristics of FSM-16 and MCM-41, which would be consistent with the observed larger adsorption capacity of cationic pigment compared with anionic pigment for these materials. The immobilized HRP on FSM-16 and MCM-41 with pore diameter 50 A showed the highest enzymatic activity in an organic toluene and thermal stability in aqueous solution at the temperature of 70 °C. The immobilized enzymes on the other mesoporous materials including large or small pore sized FSM-16 showed lower enzymatic activity in an or...
466 citations
TL;DR: An automatic reactor simulating, at laboratory scale, the performance of an industrial stirred tank reactor (STR) is described, and its utilization for evaluating theperformance of immobilized enzymes is shown.
Abstract: Eupergit® C is a carrier consisting of macroporous beads for immobilizing enzymes of industrial potential for the production of fine chemicals and pharmaceuticals. Various enzymes immobilized on Eupergit® C are reviewed in comparison with other carrier materials in terms of the operational stability of the respective biocatalysts at substrate concentrations realistic for industrial production. Other aspects of relevance in that field, such as the demand for purity of enzyme to be immobilized or type of reactor optimal for a given application, are also discussed. An automatic reactor simulating, at laboratory scale, the performance of an industrial stirred tank reactor (STR) is described, and its utilization for evaluating the performance of immobilized enzymes is shown.
423 citations
TL;DR: A novel method to achieve lipase immobilization by entrapment in chemically inert hydrophobic silica gels which are prepared by hydrolysis of alkyl‐substituted silanes in the presence of the enzyme is presented.
Abstract: The commercial application of lipases as biocatalysts for organic synthesis requires simple but efficient methods to immobilize the enzyme, yielding highly stable and active biocatalysts which are easy to recover. In this study, we present a novel method to achieve lipase immobilization by entrapment in chemically inert hydrophobic silica gels which are prepared by hydrolysis of alkyl-substituted silanes in the presence of the enzyme. A typical immobilization procedure uses: an aqueous solution of lipase; sodium fluoride as a catalyst; and additives like polyvinyl alcohol or proteins and alkoxysilane derivatives like RSi-(OMe)3 with R = alkyl, aryl, or alkoxy as gel precursors. The effect of various immobilization parameters like stoichiometric ratio of water, silane, type and amount of additive, type and amount of catalyst, and type of silane has been carefully studied. The new method is applicable for a wide variety of lipases, yielding immobilized lipases with esterification activities enhanced by a factor of up to 88, compared to the commercial enzyme powders under identical conditions. Studies on the stability of sol-gel immobilized lipases under reaction conditions or storage (dry, in aqueous or organic medium) revealed an excellent retention of enzymatic activity. The possible reasons for the increased enzyme activities are discussed. © 1996 John Wiley & Sons, Inc.
406 citations
TL;DR: A three-step methanolysis was developed by which over 95% of the oil triacylglycerols (TAG) were converted to their corresponding methyl esters (ME) by Candida antarctica lipase.
Abstract: Candida antarctica lipase is inactivated in a mixture of vegetable oil and more than 1∶2 molar equivalent of methanol against the total fatty acids. We have revealed that the inactivation was eliminated by three successive additions of 1∶3 molar equivalent of methanol and have developed a three-step methanolysis by which over 95% of the oil triacylglycerols (TAG) were converted to their corresponding methyl esters (ME). In this study, the lipase was not inactivated even though 2∶3 molar equivalent of methanol was present in a mixture of acylglycerols (AG) and 33% ME (AG/ME33). This finding led to a two-step methanolysis of the oil TAG: The first-step was conducted at 30°C for 12 h with shaking in a mixture of the oil, 1∶3 molar equivalent of methanol, and 4% immobilized lipase; the second-step reaction was done for 24 h after adding 2∶3 molar equivalent of methanol (36 h in total). The two-step methanolysis achieved more than 95% of conversion. When two-step reaction was repeated by transferring the immobilized lipase to a fresh substrate mixture, the enzyme could be used 70 cycles (105 d) without any decrease in the conversion. From the viewpoint of the industrial production of biodiesel fuel production, the two-step reaction was conducted using a reactor with impeller. However, the enzyme carrier was easily destroyed, and the lipase could be used only several times. Thus, we attempted flow reaction using a column packed with immobilized Candida lipase. Because the lipase packed in the column was drastically inactivated by feeding a mixture of AG/ME33 and 2∶3 molar equivalent of methanol, three-step flow reaction was performed using three columns packed with 3.0 g immobilized lipase. A mixture of vegetable oil and 1∶3 molar equivalent of methanol was fed into the first column at a constant flow rate of 6.0 mL/h. The eluate and 1∶3 molar equivalent of methanol were mixed and then fed into the second column at the same flow rate. The final step reaction was done by feeding a mixture of eluate from the second column and 1∶3 molar equivalent of methanol at the same flow rate. The ME content in the final-step eluate reached 93%, and the lipase could be used for 100 d without any decrease in the conversion.
361 citations
TL;DR: A novel aqueous sol−gel process to encapsulate biological molecules (such as enzymes, antibodies, and cells) that uses neutral pH and room temperature and does not generate alcohol as a byproduct is developed.
Abstract: Porous silica materials made by low-temperature sol−gel process are promising host matrixes for encapsulation of biomolecules. To date, researchers have focused on sol−gel routes using alkoxides such as tetramethyl orthosilicate (TMOS) and tetraethyl orthosilicate (TEOS) for encapsulation of biomolecules. These routes lead to formation of alcohol as a byproduct that can have a detrimental effect on the activity of entrapped biomolecules. We have developed a novel aqueous sol−gel process to encapsulate biological molecules (such as enzymes, antibodies, and cells) that uses neutral pH and room temperature and does not generate alcohol as a byproduct. The process uses sodium silicate as precursor and is carried out in two steps: preparation of a low-pH silicate sol followed by gelation at neutral pH with a suitable buffer containing biomolecules. Two enzymes widely used in biosensing applications, horseradish peroxidase (HRP) and glucose-6-phosphate dehydrogenase (G6PDH), were used to prepare enzyme-doped s...
341 citations
TL;DR: Progressive establishment of new enzyme-support attachments was showed by the progressive irreversible covalent immobilization of several subunits of multi-subunits proteins, and the final stabilization factor become 100-fold comparing soluble penicillin G acylase and optimal derivative.
337 citations
TL;DR: The immobilization of cytochrome c into the mesoporous moleclar sieves MCM-48 and SBA-15, as well as the layered niobium oxide Nb-TMS4 (Niobium Transition Metal Oxide Molecular Sieve) is described.
Abstract: The immobilization of cytochrome c into the mesoporous moleclar sieves MCM-48 (Mobil Composition of Matter) and SBA-15 (Santa Barbara Amorphous), as well as the layered niobium oxide Nb-TMS4 (Niobium Transition Metal Oxide Molecular Sieve) is described. The cytochrome c loading was influenced by the molecular sieve structure, i.e., one-dimensional vs. three-dimensional. Cytochrome c immobilization was accomplished by absorption into the molecular sieves followed by silylation of the pore openings. The molecular sieves containing cytochrome c were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), UV/Vis, and FT-IR spectroscopy. The protein was found to be stable in the molecular sieve under conditions that would denature the protein in solution. The immobilized cytochrome c retained its redox activity following immobilization for several months as demonstrated by cyclic voltammetry (CV).
237 citations
TL;DR: Polyethylenimine-support composites were also very suitable to perform very strong and nondistorting reversible immobilization of industrial enzymes, and DAAO, a dimeric enzyme, seems to be due to the involvement of both enzyme subunits in the ionic adsorption.
Abstract: New tailor-made anionic exchange resins have been prepared, based on films of large polyethylenimine polymers (e.g., MW 25,000) completely coating, via covalent immobilization, the surface of different porous supports (agarose, silica, polymeric resins). Most proteins contained in crude extracts from different sources have been very strongly adsorbed on them. Ionic exchange properties of such composites strongly depend on the size of polyethylenimine polymers as well as on the exact conditions of the covalent coating of the solids with the polymer. On the contrary, similar coating protocols yield similar matrices by using different porous supports as starting material. For example, 77% of all proteins contained in crude extracts from Escherichia coli were adsorbed, at low ionic strength, on the best matrices, and less than 15% of the adsorbed proteins were eluted from the support in the presence of 0.3 M NaCl. Under these conditions, 100% of the adsorbed proteins were eluted from conventional DEAE supports. Such polyethylenimine-support composites were also very suitable to perform very strong and nondistorting reversible immobilization of industrial enzymes. For example, lipase from Candida rugosa (CRL), beta-galactosidase from Aspergillus oryzae and D-amino acid oxidase (DAAO) from Rhodotorula gracilis, were adsorbed on such matrices in a few minutes at pH 7.0 and 4 degrees C. Immobilized enzymes preserved 100% of catalytic activity and remained fully immobilized in 0.2 M NaCl. In addition to that, CRL and DAAO were highly stabilized upon immobilization. Stabilization of DAAO, a dimeric enzyme, seems to be due to the involvement of both enzyme subunits in the ionic adsorption.
235 citations
TL;DR: The effects of the pretreatment of immobilized Candida antarctica lipase enzyme (Novozym 435) on methanolysis for biodiesel fuel production were investigated and a procedure for the stepwise addition of methanol to the reaction mixture so as to maintain the desired meethanol content was determined.
Book•
01 Jan 2000
TL;DR: This work describes biomaterials that communicate antigen-antibody assembling of enzyme monomolecular layers and multilayers on electrodes as well as electrochemistry and electroenzymology in layered films containing proteins.
Abstract: Part 1 Protein adsorption on a solid surface: protein adsorption on solid/liquid interface. Part 2 Surface covalent immobilization of proteins: immobilization of protein monolayers on planar solid supports. Part 3 Protein monolayer formation at liquid interface and its transfer on solid substrate: quantitative detection of protein binding to the lipid membrane surface by using quartz crystal microbalance ordering by capillary forces Langmuir-Blodgett multilayers of proteins. Part 4 Electrostatic layer-by-layer assembly via alternate adsorption of proteins and polyions: electrostatic layer-by-layer assembly of proteins and polyions sequential catalysis in organized multienzyme films fabrication of immunoglobulin mono- and multilayers - their applications for immunosensors immobilized enzyme multilayers -the way for assembly of microreactors and biosensors ordered multilayer assemblies - albumin/heparin for biocompatible coating and monoclonal antibodies for optical immunosensors. Part 5 Multilayer assembly with biospecific interaction: biomaterials that communicate antigen-antibody assembling of enzyme monomolecular layers and multilayers on electrodes. Part 6 Electrochemistry in ordered protein layers: electrochemistry and electroenzymology in layered films containing proteins. Part 7 Patterning of protein layers - two dimensional patterning of proteins.
TL;DR: A laccase, the only ligninolytic enzyme produced by the basidiomycete Pleurotus ostreatus strain RK 36 was purified to homogeneity and characterized and Benzoate was found to stabilize the enzyme during the immobilization process.
TL;DR: Overall the enzyme‐modified rods performed substantially better than the corresponding beads, and the performance of the molded supports as enzymatic reactors or as chromatographic media benefits greatly from the enhanced mass transfer that is characteristic of the modeled rod at high flow rates.
Abstract: Trypsin immobilization onto continuous "molded" rods of porous poly(glycidyl methacrylate-co-ethylene dimethacrylate) and some applications of the conjugate have been studied. The rods polymerized within a tubular mold (chromatographic column), were treated in situ with ethylenediamine, activated with glutaraldehyde and finally modified with trypsin. The performance of the trypsin-modified rods was evaluated and compared to that of poly(glycidyl methacrylate-co-ethylene dimethacrylate) beads, modified with the same enzyme. Overall the enzyme-modified rods performed substantially better than the corresponding beads. In particular, the performance of the molded supports as enzymatic reactors or as chromatographic media benefits greatly from the enhanced mass transfer that is characteristic of the molded rod at high flow rates. (c) 1996 John Wiley & Sons, Inc.
TL;DR: A review of recent developments in immobilized enzyme use in medicine and a brief overview of the evolution of the biosensor and bioreactor technology are presented.
TL;DR: It was discovered that horseradish peroxidase was selectively adsorbed on magnetite, and the immobilization resulted in a 20-fold purification rate for crude enzyme.
Abstract: Immobilization of horseradish peroxidase on magnetite and removal of chlorophenols using immobilized enzyme were investigated. Immobilization by physical adsorption on magnetite was much more effective than that by the crosslinking method, and the enzyme was found to be immobilized at 100% of retained activity. In addition, it was discovered that horseradish peroxidase was selectively adsorbed on magnetite, and the immobilization resulted in a 20-fold purification rate for crude enzyme. When immobilized peroxidase was used to treat a solution containing various chlorophenols, p-chlorophenol, 2,4-dichlorophenol, 2,4,5-trichlorophenol, 2,4,6-trichlorophenol, 2,3,4,6-tetrachlorophenol, and pentachlorophenol, each chlorophenol was almost 100% removed, and also the removal of total organic carbon (TOC) and adsorbable organic halogen (AOX) reached more than 90%, respectively. However, in the case of soluble peroxidase, complete removal of each chlorophenol could not be attained, and in particular, the removal of 2,4,5-trichlorophenol was the lowest, with a removal rate of only 36%.
TL;DR: It was found that an increase in the concentration of sodium alginate and calcium chloride gives rise to a reduction in the enzyme leakage, which was verified when glucose oxidase (GOD) diffusion percentages were compared in capsules with thicknesses of the same order of magnitude but obtained under different experimental conditions.
TL;DR: A hydrogen peroxide biosensor was fabricated by coating a sol-gel-peroxidase layer onto a Nafion-methylene green modified electrode, and the sensor achieved 95% of the steady-state current within 20 s.
TL;DR: The cyclic voltammograms obtained from the Au electrodes modified with the GOx/dendrimer multilayers revealed that bioelectrocatalytic response is directly correlated to the number of deposited bilayers, that is, to the amount of active enzyme immobilized on the Au electrode surface.
Abstract: A new approach to construct a multilayered enzyme film on the Au surface for use as a biosensing interface is described. The film was prepared by alternate layer-by-layer depositions of G4 poly(amidoamine) dendrimers and periodate-oxidized glucose oxidase (GOx). The cyclic voltammograms obtained from the Au electrodes modified with the GOx/dendrimer multilayers revealed that bioelectrocatalytic response is directly correlated to the number of deposited bilayers, that is, to the amount of active enzyme immobilized on the Au electrode surface. From the analysis of voltammetric signals, the coverage of active enzyme per GOx/dendrimer bilayer during the multilayer-forming steps was estimated, which demonstrates that the multilayer is constructed in a spatially ordered manner. Also, with the ellipsometric measurements, a linear increment of the film thickness was registered, supporting the formation of the proposed multilayered structure. The E5D5 electrode showed the sensitivity of 14.7 μA·mM-1 glucose·cm-2 a...
TL;DR: This review discusses the debittering enzyme naringinase and its essential role in the commercial processing of citrus fruit juice and its applications in other areas are identified.
TL;DR: In this article, the properties of active enzymatic complexes were investigated, which were formed by the interaction of acid phosphatase with clays (montmorillonite and Al hydroxide), tannic acid and organo-mineral aggregates.
Abstract: The properties of synthetic active enzymatic complexes, simulating those usually present in soil environment, were investigated. Complexes were formed by the interaction of acid phosphatase with clays (montmorillonite and Al hydroxide), tannic acid and organo-mineral aggregates, obtained by mixing tannate, OH-Al species and/or montmorillonite. Immobilized acid phosphatase showed catalytic features quite different from those of the free enzyme. The presence of OH-Al species in the matrix generally resulted in an improvement of some enzymatic properties. A gain in activity of about 45 and 55% was observed for the complexes acid phosphatase–tannate–OH-Al species after thermal deactivation at 60°C and 2 h of exposure to proteinase K. High residual activities ranging from 17 to 61% and from 28 to 57% of the initial one were measured for complexes of the enzyme with inorganic and organic/organo-mineral matrices, respectively. In contrast, the association with a pure constituent such as montmorillonite and/or tannic acid gave rise to an immobilized enzyme, displaying a completely different catalytic behaviour. Compared to the free enzyme, acid phosphatase–montmorillonite and acid phosphatase–tannate complexes had a different pH-activity dependence and a higher and lower sensitivity to temperature and proteolysis, respectively.
TL;DR: In this paper, a new enzymatic amperometric biosensor for hydrogen peroxide (H2O2) was developed via an easy and effective enzyme immobilization method using chitosan film crosslinked with glutaraldehyde.
Abstract: A new enzymatic amperometric biosensor for hydrogen peroxide
(H2O2) was developed via an easy and
effective enzyme immobilization method using chitosan film crosslinked with
glutaraldehyde. Horseradish peroxidase was immobilized on the surface of a
carbon paste electrode. Hexacyanoferrate(II) was present in the
solution as a mediator. The biosensor exhibited a relatively fast response
of less than 10 s and produced currents linearly related to the
H2O2 concentration in the range of 4.7 ×
10−5 to 2 × 10−3 M. The biosensor
had very good stability as it retained ca. 85% activity after 30 d
of storage in a phosphate buffer at 4 °C. The effect of the chitosan
film thickness and various operational parameters were optimized. The
applicability of this biosensor was demonstrated with the analysis of real
samples and the results obtained by this biosensor corroborated well with
the classical iodometric titration method.
TL;DR: Assay results show that mesoporous MCM-41 is a more effective support for the immobilization of Penicillin Acylase (PA) than many of other supports due to its structural and surface characteristics.
Abstract: Mesoporous MCM-41 having well ordered long-range structure, large pore diameters, narrow pore-size distribution, high pore volume and specific surface area has been synthesized. The surface of MCM-41 has an abundance of weakly acidic hydroxyl groups. Assay results show that MCM-41 is a more effective support for the immobilization of Penicillin Acylase (PA) than many of other supports due to its structural and surface characteristics. PA can be immobilized on MCM-41 through either direct immobilization or covalent coupling. The former gives higher activity of IME than the later. In the direct immobilization, PA molecules are immobilized on MCM-41 through the hydrogen-bonded interaction between hydroxyl groups of MCM-41 and carbonyl or amino groups in the PA molecule.
TL;DR: In this paper, a peroxidase biosensor was fabricated by simply dropping sor-gel-peroxide mixture onto glassy carbon electrode surface, and the response time was about 10 s; the linear range was up to 3.4 mM with a detection limit of 5 x 10(-7) M. The sensor exhibited high sensitivity (15 mu A mM(-1)) and good long-term stability.
TL;DR: The kinetic model of the hydrolysis of lactose with a beta-galactosidase from Kluyveromyces fragilis immobilized on a commercial silica-alumina (KA-3, from Südchemie) has been determined and a Michaelis-Menten model with a competitive inhibition by galactose has been selected.
TL;DR: In this paper, Lactate oxidase (LOD) and lactate dehydrogenase (LDH) were co-immobilized on electrochemically prepared polyaniline (PANI) films by physical adsorption.
TL;DR: In addition to chitinase/lysozyme, Pseudomonas aeruginosa K-187 produced a protease useful for the deproteinization of shrimp and crab shell wastes, which has resulted in a 67% protein removal.
TL;DR: In this paper, two synthetically useful glycosidases, the β-galactosidase from Bacillus circulans and an α-galacticase from Aspergillus oryzae have been immobilized on Eupergit C. They retain high catalytic activity and show increased thermal stability compared with the free enzymes.
TL;DR: A thermostable lipase produced by a thermophilic Bacillus sp.
Abstract: A thermostable lipase produced by a thermophilic Bacillus sp. J33 was purified to 175-fold with 15.6% recovery by ammonium sulphate and Phenyl Sepharose column chromatography. The enzyme is a monomeric protein having molecular weight of 45 kDa. It hydrolyzes triolein at all positions. The fatty acid specificity of lipase is broad with little preference for C12 and C4. The Km and Vmax for lipase with pNP-laurate as substrate was calculated to be 2.5 mM and 0.4 μM min-1 ml-1 respectively. The immobilized enzyme was stable for 12 h at 60°C. Polyhydric alcohols such as ethylene glycol (2.5 M), sorbitol (2.5 M) and glycerol (2.5 M) were used as thermostabilizers. Lipase acquired a remarkable stability, since no deactivation occurred at 70°C for 150 min in the presence of additives.
TL;DR: A second order response function was developed based on Box-Behnken design of experiments, which indicated optimum conditions for maximum esterification and increased with both E/S ratio and time and decreased with alcohol (acid) concentration.