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Immobilized Enzyme (immobilized + enzyme)
Selected AbstractsIntegrated Enzymatic Synthesis and Adsorption of Isomaltose in a Multiphase Fluidized Bed ReactorENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 5 2006M. Ergezinger Abstract Dextransucrase catalyzes the formation of dextran, but also of numerous oligosaccharides from sucrose and different acceptors, if appropriate conditions are chosen. A process on a technical scale with immobilized enzyme was established to produce isomaltose, a disaccharide of industrial interest. Isomaltose is also a reactant for dextransucrase and has to be quickly taken out of the reaction solution. This was realized by integrated adsorption of isomaltose on zeolites. In the case of biotransformation the reactor works with a fluidized bed of immobilized enzyme and the in situ separation is realized with a suspension flow of adsorbent. This process was investigated experimentally and theoretically. With a design model consisting of hydrodynamics, kinetics of enzymatic synthesis, and thermodynamics of adsorption, a comparison was made between experimental and calculated data. [source] Functionalized Multi-Wall Carbon Nanotubes for Lipase Immobilization,ADVANCED ENGINEERING MATERIALS, Issue 5 2010I. V. Pavlidis Abstract We examine the immobilization of lipase B from Candida antarctica on functionalized multi-wall carbon nanotubes (MWCNTs) through physical adsorption. MWCNTs functionalized with carboxyl-, amine- and ester- terminal groups on their surface are used as immobilization carriers. Dispersion of the nanotubes and the immobilization procedure take place in aqueous and low-water media. High enzyme loadings are attained, up to 25% of the weight of the carbon nanotubes. These novel biomaterials are characterized though FT-IR and Raman spectroscopy. The MWCNT,lipase bioconjugates exhibit high catalytic activity and increased storage and operational stability. The biomaterials retain more than 55% of their initial activity after 6 months at 4,°C, while they retain approximately 25% of their initial activity after 30 d of incubation in hexane at 60,°C. The catalytic behaviour of the immobilized enzyme depends on the terminal group of the carbon nanotubes, the concentration of the enzyme and the immobilization method employed. [source] Bottom-Up Synthesis of Biologically Active Multilayer Films Using Inkjet-Printed TemplatesADVANCED FUNCTIONAL MATERIALS, Issue 9 2010Joshua D. Swartz Abstract As a non-invasive, rapid prototyping technique, piezoelectric inkjet printing using the Dimatix Materials Printer (DMP) is incorporated to template 2D biologically active surfaces. In these studies, a bioinspired ink is synthesized and printed directly onto gold-coated silicon nitride substrates and into polymer-coated 96-well plates. Once deposited on a surface, these patterns are reacted with varying concentrations of a model enzyme glucose oxidase in the presence of a silica precursor, monosilicic acid. The reaction mechanism and order of reactant products within and along the patterns are shown to directly affect the integrity and overall microstructure of the biologically active films. Using profilometry measurements and scanning electron microscopy, a biologically active platform is optimized without significantly compromising the activity of the enzyme. In fact, enzyme activity, constrained within a thin film, is reported for the first time over variable reaction parameters. When compared to the enzyme free in solution, the immobilized enzyme is 25.9% active, where nearly 100% of the activity is retained after repeated usage. [source] Efficient Biocatalytic Cleavage and Recovery of Organic Substrates Supported on Soluble PolymersADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 6 2007Dario Pasini Abstract The applicability of novel solution-phase supports in combination with enzymes for biocatalytic transformations is reported. Ex novo designed styrene-based copolymers, bearing a phenylacetic residue in variable loadings and linked as a pendant group to the macromolecular backbone, through a spacer of variable length, have been synthesized and characterized. These derivatives are compatible and can be used as soluble supports in combination with immobilized penicillin G acylase (PGA , EC 3.5.1.11) for the biocatalytic cleavage of the covalently anchored organic substrate in quantitative yields, in water or water/dimethylformamide solvent mixtures, with recovery of the immobilized enzyme with negligible losses in activity. [source] Covalent immobilization of ,-galactosidase on carrageenan coated with chitosanJOURNAL OF APPLIED POLYMER SCIENCE, Issue 1 2009Magdy M.M. Elnashar Abstract ,-Galactosidase was covalently immobilized to carrageenan coated with chitosan for the hydrolysis of lactose. The chitosan-carrageenan polyelectrolyte interaction was found to be dependent on the chitosan pH. At pH 4, the chitosan reached its maximum binding of 28.5% (w/w) where the chitosan surface density was 4.8 mg chitosan/cm2 g of carrageenan gel disks, using Muzzarelli method. Glutaraldehyde was used as a mediator to incorporate new functionality, aldehydic carbonyl group, to the bio-polymers for covalent attachment of ,-galactosidase. The enzyme was covalently immobilized to the biopolymer at a concentration of 2.73 mg protein per g of wet gel. FTIR proved the incorporation of the aldehydic carbonyl group to the carrageenan coated with chitosan at 1720 cm,1. The optimum time for enzyme immobilization was found to be 16 h, after which a plateau was reached. The enzyme loading increased from 2.65 U/g (control gel) to 10.92 U/g gel using the covalent technique. The gel's modification has shown to improve the carrageenan gel thermal stability as well as the immobilized enzyme. For example, the carrageenan gel treated with chitosan showed an outstanding thermal stability at 95°C compared with 35°C for the untreated carrageenan gel. Similarly, the immobilization process shifted the enzyme's optimum temperature from 50°C for the free enzyme towards a wider temperature range 45,55 °C indicating that the enzyme structure is strengthened by immobilization. In brief, the newly developed immobilization method is simple; the carrier is cheap, yet effective and can be used for the immobilization of other enzymes. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009 [source] Improvement of enantioselectivity and stability of Klebsiella oxytoca hydrolase immobilized on Eupergit C 250LJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 11 2008Pei-Yun Wang Abstract BACKGROUND: A simple procedure was employed to covalently immobilize a Klebsiella oxytoca hydrolase (SNSM-87) onto epoxy-activated supports of Eupergit C 250L via multipoint covalent attachment. The resultant biocatalyst was explored for the hydrolytic resolution of a variety of (R,S)-2-hydroxycarboxylic acid ethyl esters. RESULTS: With the hydrolytic resolution of (R,S)-ethyl mandelate in biphasic media as the model system, optimal conditions of 55 °C, pH 6 buffer and isooctane as the organic phase were selected for improving the enzyme stability (activity retained from 10% to 50% at 96 h) and enantioselectivity (VSVR,1 value enhanced from 44 to 319) in comparison to the performance of free enzyme. Moreover, the immobilized enzyme retained its activity and enantioselectivity after eight cycles of hydrolysis at 55 °C. When applying the resolution process to other (R,S)-2-hydroxycarboxylic acid ethyl esters, 2.4- to 4.0-fold enhancements of the enantioselectivity in general were obtainable. CONCLUSIONS: The enantioselectivity enhancement, good reusability and easy recovery after reaction indicate that the immobilized SNSM-87 may have the potential as an industrial biocatalyst for the preparation of optically pure 2-hydroxycarboxylic acids. Copyright © 2008 Society of Chemical Industry [source] Stabilization of penicillin V acylase from Streptomyces lavendulae by covalent immobilizationJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 5 2001Jesús Torres-Bacete Abstract Penicillin,V acylase from the actinomycete Streptomyces lavendulae ATCC 13664 has been immobilized to epoxy-activated acrylic beads (Eupergit C®) by covalent binding. Further linkage of bovine serum albumin after enzyme immobilization was carried out in order to remove the remaining oxirane groups of the support. The obtained immobilized biocatalyst displayed double exponential deactivation kinetics at temperatures below 55,°C, while the native enzyme followed single exponential decay at the same temperatures. We concluded that soluble penicillin acylase was deactivated in one step, whereas the immobilized enzyme showed an enzymatic intermediate state which is highly thermostable. As a consequence of the immobilization process, the enzyme displayed a 10-fold increase in its half-life at 40,°C. At this temperature, the enzymatic intermediate state was progressively destabilized as the pH of the medium was increased. Thus, the optimum pH range for the immobilized enzyme preparation was established as being from 7.0 to 8.0. Higher pH values led to quicker enzyme deactivation. © 2001 Society of Chemical Industry [source] COVALENT IMMOBILIZATION OF INVERTASE ON CHEMICALLY ACTIVATED POLY (STYRENE-2-HYDROXYETHYL METHACRYLATE) MICROBEADSJOURNAL OF FOOD BIOCHEMISTRY, Issue 3 2008HAYDAR ALTINOK ABSTRACT A carrier for invertase enzyme was synthesized from styrene (S) and 2- hydroxyethyl methacrylate (HEMA) in the form of microbeads. These poly (styrene-2-hydroxyethyl methacrylate), P(S-HEMA) microbeads were activated by epichlorohydrin (ECH) treatment for covalent immobilization. The free and immobilized invertase were assayed in the hydrolysis of sucrose to glucose, and the obtained results were compared. The optimum pH was 4.5 for free and 5.5 for immobilized invertase. The optimum temperature of invertase shifted from 45C to 55C upon immobilization. For free and immobilized enzymes, kinetic parameters were calculated as 4.1 × 10,3 mol L,1and 9.2 × 10,3 mol L,1for Km, and 6.6 × 10,2 mol L,1 min,1and 4.1 × 10,1 mol L,1 min,1for Vmax, respectively. After 1 month of storage at 4C, free enzyme retained 36% of its initial activity, while for the ECH-activated P(S-HEMA) immobilized enzyme, P(S-HEMA)-E, this value was observed as 67%. In repeated batch use, i.e., 20 times in 3 days, 78% retention of the initial activity was observed for P(S-HEMA)-E system. PRACTICAL APPLICATIONS Immobilization of enzymes are very important for many industrial applications, e.g., food, medicine, pharmacology, etc. Invertase converts sucrose to glucose and fructose, which have wide applications in food industry especially as sweeteners. Glucose,fructose mixture has much lower crystallinity compared to sucrose and therefore used in the production of noncrystallizing jams and creams. They are also used as liquid sweeteners. Immobilization enables repeated use, provides significant reduction in the operation costs, facilitates easy separation and speeds up recovery of enzyme and extends the stability of enzyme by protecting the active material from deactivation. Industrial application of immobilized invertase may decrease the production cost of glucose,fructose mixture because it could be used repeatedly for long periods. Although invertase is not a very expensive enzyme, the technique can also be applied to expensive ones for biotechnological productions. [source] Determination of sulphite using an immobilized enzyme with ,ow injection chemiluminescence detectionLUMINESCENCE: THE JOURNAL OF BIOLOGICAL AND CHEMICAL LUMINESCENCE, Issue 1 2004M. Yaqoob Abstract A ,ow injection method is reported for the determination of sulphite-based on chemiluminescent detection. Hydro-gen peroxide is produced from sulphite using on-line covalently bound immobilized sulphite oxidase packed in a mini-column, which was mixed downstream and detected via cobalt(II)-catalysed chemiluminescent oxidation of luminol. The limit of detection (2 × standard deviation of the blank) was 1 × 10,3 mmol/L with sample throughput 60 h,1. The calibration data was linear over the range of 0.2,1.0 mmol/L with relative standard deviation (n = 4) in the range 0.9,2.0%. Copyright © 2004 John Wiley & Sons, Ltd. [source] The Properties of Covalently Immobilized Trypsin on Soap-Free P(MMA-EA-AA) Latex ParticlesMACROMOLECULAR BIOSCIENCE, Issue 4 2005Kai Kang Abstract Summary: The covalent immobilization of trypsin onto poly[(methyl methacrylate)- co -(ethyl acrylate)- co -(acrylic acid)] latex particles, produced by a soap-free emulsion polymerization technique, was carried out using the carbodiimide method. The catalytic properties and kinetic parameters, as well as the stability of the immobilized enzyme were compared to those of the free enzyme. Results showed that the optimum temperature and pH for the immobilized trypsin in the hydrolysis of casein were 55,°C and 8.5, both of which were higher than that of the free form. It was found that Km (Michaelis constant) was 45.7 mg,·,ml,1 and Vmax (maximal reaction rate) was 793.0 ,g,·,min,1 for immobilized trypsin, compared to a Km of 30.0 mg,·,ml,1 and a Vmax of 5,467.5 ,g,·,min,1 for free trypsin. The immobilized trypsin exhibited much better thermal and chemical stabilities than its free counterpart and maintained over 63% of its initial activity after reusing ten times. TEM photograph of latex particles after trypsin immobilization. [source] Stability of ,-galactosidase immobilized on composite microspheres of artemisia seed gum and chitosanPOLYMER COMPOSITES, Issue 1 2008Ji Zhang In this work, composite microspheres were prepared by using artemisia seed gum and chitosan as a source. The composite microspheres have activated aldehyde groups by using glutaraldehyde. ,-Galactosidase was covalently bound on these activated microspheres. The properties of the immobilized enzyme were investigated and compared with those of the free enzyme, for which o -nitrophenol ,- D -galactopyranoside (ONPG) was chosen as a substrate. The results showed that the pH and thermal stability of the immobilized ,-galactosidase were higher than those of the soluble one. Apart from these, the Michaelis constant Km was evaluated for the immobilized ,-galactosidase and the soluble enzyme. The immobilized ,-galactosidase exhibited better environmental adaptability and reusability than the soluble one. POLYM. COMPOS., 29:9,14, 2008. © 2007 Society of Plastics Engineers [source] In this issue: Biotechnology Journal 8/2010BIOTECHNOLOGY JOURNAL, Issue 8 2010Article first published online: 12 AUG 2010 Biocatalyst microemulsions Pavlidis et al., Biotechnol. J. 2010, 5, 805,812 Enzymes maintain their catalytic activity when hosted in aqueous nanodroplets like reverse micelles. Researchers from Ioannina, Greece, propose the use of water-in-ionic liquid microemulsionbased organogels (w/IL MBGs) as novel supports for the immobilization of lipase B from Candida antarctica and lipase from Chromobacterium viscosum. These novel lipase-containing w/IL MBGs can be effectively used as solid phase biocatalysts in various polar and non-polar organic solvents or ILs, exhibiting up to 4.4-fold higher esterification activity compared to water-in-oil microemulsion-based organogels. The immobilized lipases retain their activity for several hours at 70°C, while their half life time is up to 25-fold higher compared to that observed in w/IL microemulsions Biocatalyst cryogelation Bieler et al., Biotechnol. J. 2010, 5, 881,885 Entrapment of biocatalysts in hydrogel beads allows stable operation in otherwise deteriorating solvents. Doing this by cryogelation is a gentle method to extend the scope of biocatalysis. To foster the use of this versatile method, researchers from Aachen, Germany, devised an automated injector for the production of PVA/PEG-enzyme immobilisates. The device consists of a thermostated reservoir connected to a programmable injector nozzle and an agitated receiving bath for the droplets. This lab-scale production unit yields up to 1500 beads with immobilized enzyme per minute with a narrow size distribution and good roundness. Biocatalyst membrane reactor Lyagin et al., Biotechnol. J. 2010, 5, 813,821 Screening of biocatalysts, substrates or conditions in the early stages of bioprocess development requires an enormous number of experiments and is a tedious, expensive and time-consuming task. Currently available screening systems can only be operated in batch or fed-batch mode, which can lead to severe misinterpretations of screening results. Researchers from Berlin, Germany, now developed a novel screening system that enables continuous feeding of substrates and continuous removal of products. A prototype based on the membrane reactor concept was designed and operated for a model reaction, the hydrolysis of cellulose. [source] Automated lab-scale production of PVA/PEG-enzyme immobilisatesBIOTECHNOLOGY JOURNAL, Issue 8 2010Nora C. Bieler Abstract Entrapment of biocatalysts by cryogelation is a gentle method to extend the scope of biocatalysis. To foster the use of this versatile method we devised an automated injector for the production of PVA/PEG beads. The device consists of a thermostated reservoir connected to a programmable injector nozzle and an agitated receiving bath for the droplets. This lab-scale production unit yields up to 1500 beads with immobilized enzyme per minute with a narrow size distribution and good roundness. [source] Immobilization of a thermostable ,-amylase by covalent binding to an alginate matrix increases high temperature usabilityBIOTECHNOLOGY PROGRESS, Issue 2 2009Boon L. Tee Abstract Thermostable ,-amylase was covalently bound to calcium alginate matrix to be used for starch hydrolysis at liquefaction temperature of 95°C. 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride (EDAC) was used as crosslinker. EDAC reacts with the carboxylate groups on the calcium alginate matrix and the amine groups of the enzyme. Ethylenediamine tetraacetic acid (EDTA) treatment was applied to increase the number of available carboxylate groups on the calcium alginate matrix for EDAC binding. After the immobilization was completed, the beads were treated with 0.1 M calcium chloride solution to reinstate the bead mechanical strength. Enzyme loading efficiency, activity, and reusability of the immobilized ,-amylase were investigated. Covalently bound thermostable ,-amylase to calcium alginate produced a total of 53 g of starch degradation/mg of bound protein after seven consecutive starch hydrolysis cycles of 10 min each at 95°C in a stirred batch reactor. The free and covalently bound ,-amylase had maximum activity at pH 5.5 and 6.0, respectively. The Michaelis-Menten constant (Km) of the immobilized enzyme (0.98 mg/mL) was 2.5 times greater than that of the free enzyme (0.40 mg/mL). The maximum reaction rate (Vmax) of immobilized and free enzyme were determined to be 10.4-mg starch degraded/mL min mg bound protein and 25.7-mg starch degraded/mL min mg protein, respectively. The high cumulative activity and seven successive reuses obtained at liquefaction temperature make the covalently bound thermostable ,-amylase to calcium alginate matrix, a promising candidate for use in industrial starch hydrolysis process. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Enhancing the Reusability of Endoglucanase-Gold Nanoparticle Bioconjugates by Tethering to Polyurethane MicrospheresBIOTECHNOLOGY PROGRESS, Issue 6 2004Sumant Phadtare The synthesis of polyurethane microsphere-gold nanoparticle "core-shell" structures and their use in the immobilization of the enzyme endoglucanase are described. Assembly of gold nanoparticles on the surface of polymer microspheres occurs through interaction of the nitrogens in the polymer with the nanoparticles, thereby precluding the need for modifying the polymer microspheres to enable such nanoparticle binding. Endoglucanse could thereafter be bound to the gold nanoparticles decorating the polyurethane microspheres, leading to a highly stable biocatalyst with excellent reuse characteristics. The immobilized enzyme retains its biocatalytic activity and exhibits improved thermal stability relative to free enzyme in solution. The high surface area of the host gold nanoparticles renders the immobilized enzyme "quasi free", while at the same time retaining advantages of immobilization such as ease of reuse, enhanced temporal and thermal stability, etc. [source] Characteristics of Immobilized Lipase on Hydrophobic Superparamagnetic Microspheres To Catalyze EsterificationBIOTECHNOLOGY PROGRESS, Issue 2 2004Zheng Guo A novel immobilized lipase (from Candida rugosa) on hydrophobic and superparamagnetic microspheres was prepared and used as a biocatalyst to catalyze esterification reactions in diverse solvents and reaction systems. The results showed that the immobilized lipase had over 2-fold higher activities in higher log P value solvents. An exponential increase of lipase activity against log P of two miscible solvent mixtures was observed for the first time. Both free and immobilized lipase achieved its maximum activity at the range of water activity ( aw) 0.5,0.8 or higher. At aw 0.6, the immobilized lipase exhibited markedly higher activities in heptane and a solvent-free system than did the native lipase. In multicompetitive reactions, the alcohol specificity of the lipase showed a strong chain-length dependency, and the immobilized enzyme exhibited more preference for a longer-chain alcohol, which is different from previous reports. The immobilized lipase showed higher specificities for butyric acid and the medium-chain-length fatty acids (C8,C12). Then, the immobilized lipase was extended to solvent-free synthesis of glycerides from glycerol and fatty acids. Recovered by magnetic separation, the immobilized lipase exhibited good reusability in repeated batch reaction, indicating its promising feature for biotechnology application. [source] Enzymatic Synthesis of 3,-Hydroxyacetaminophen Catalyzed by TyrosinaseBIOTECHNOLOGY PROGRESS, Issue 6 2003Edelmira Valero 3,-Hydroxyacetaminophen, a catechol metabolite of N -acetyl- p -aminophenol (acetaminophen) and N -acetyl- m -aminophenol (a structural analogue of acetaminophen and considered as a possible alternative because it is not hepatotoxic), is enzymatically synthesized for the first time using mushroom tyrosinase. Although reported to be weakly hepatotoxic in vivo, this catechol derivative of acetaminophen is not commercially available. This compound was obtained from its monophenolic precursor, acetaminophen, using the enzyme tyrosinase in the presence of an excess of ascorbic acid, thus reducing back the o -quinone product of catalytic activity to the catechol acetaminophen derivative. A mathematical model of the system is proposed, which is also applicable to the tyrosinase-mediated synthesis of any o -diphenolic compound from its corresponding monophenol. This synthesis procedure is continuous, easy to perform and control, and adaptable to a bioreactor with the immobilized enzyme for industrial purposes in a nonpolluting way. [source] A Feasible Enzymatic Process for d -Tagatose Production by an Immobilized Thermostable l -Arabinose Isomerase in a Packed-Bed BioreactorBIOTECHNOLOGY PROGRESS, Issue 2 2003Hye-Jung Kim To develop a feasible enzymatic process for d -tagatose production, a thermostable l -arabinose isomerase, Gali152, was immobilized in alginate, and the galactose isomerization reaction conditions were optimized. The pH and temperature for the maximal galactose isomerization reaction were pH 8.0 and 65 °C in the immobilized enzyme system and pH 7.5 and 60 °C in the free enzyme system. The presence of manganese ion enhanced galactose isomerization to tagatose in both the free and immobilized enzyme systems. The immobilized enzyme was more stable than the free enzyme at the same pH and temperature. Under stable conditions of pH 8.0 and 60 °C, the immobilized enzyme produced 58 g/L of tagatose from 100 g/L galactose in 90 h by batch reaction, whereas the free enzyme produced 37 g/L tagatose due to its lower stability. A packed-bed bioreactor with immobilized Gali152 in alginate beads produced 50 g/L tagatose from 100 g/L galactose in 168 h, with a productivity of 13.3 (g of tagatose)/(L-reactor·h) in continuous mode. The bioreactor produced 230 g/L tagatose from 500 g/L galactose in continuous recycling mode, with a productivity of 9.6 g/(L·h) and a conversion yield of 46%. [source] Immobilization of ,-Galactosidase on Fibrous Matrix by Polyethyleneimine for Production of Galacto-Oligosaccharides from LactoseBIOTECHNOLOGY PROGRESS, Issue 2 2002Nedim Albayrak The production of galacto-oligosaccharides (GOS) from lactose by Aspergillus oryzae ,-galactosidase immobilized on cotton cloth was studied. A novel method of enzyme immobilization involving PEI,enzyme aggregate formation and growth of aggregates on individual fibrils of cotton cloth leading to multilayer immobilization of the enzyme was developed. A large amount of enzyme was immobilized (250 mg/g support) with about 90,95% efficiency. A maximum GOS production of 25,26% (w/w) was achieved at near 50% lactose conversion from 400 g/L of lactose at pH 4.5 and 40 °C. Tri- and tetrasaccharides were the major types of GOS formed, accounting for about 70% and 25% of the total GOS produced in the reactions, respectively. Temperature and pH affected not only the reaction rate but also GOS yield to some extend. A reaction pH of 6.0 increased GOS yield by as much as 10% compared with that of pH 4.5 while decreased the reaction rate of immobilized enzyme. The cotton cloth as the support matrix for enzyme immobilization did not affect the GOS formation characteristics of the enzyme under the same reaction conditions, suggesting diffusion limitation was negligible in the packed bed reactor and the enzyme carrier. Increase in the thermal stability of PEI-immobilized enzyme was also observed. The half-life for the immobilized enzyme on cotton cloth was close to 1 year at 40 °C and 21 days at 50 °C. Stable, continuous operation in a plug-flow reactor was demonstrated for about 3 days without any apparent problem. A maximum GOS production of 26% (w/w) of total sugars was attained at 50% lactose conversion with a feed containing 400 g/L of lactose at pH 4.5 and 40 °C. The corresponding reactor productivity was 6 kg/L/h, which is several-hundred-fold higher than those previously reported. [source] Protease Immobilization on ,-Fe2O3/Fe3O4 Magnetic Nanoparticles for the Synthesis of Oligopeptides in Organic SolventsCHEMISTRY - AN ASIAN JOURNAL, Issue 6 2010Bao-juan Xin Abstract The use of nanobiocatalysts, with the combination of nanotechnology and biotechnology, is considered as an exciting and rapidly emerging area. The use of iron oxide magnetic nanoparticles, as enzyme immobilization carriers, has drawn great attention because of their unique properties, such as controllable particle size, large surface area, modifiable surface, and easy recovery. In this study, various ,-Fe2O3/Fe3O4 magnetic nanoparticles with immobilized proteases were successfully prepared by three different immobilization strategies including A),direct binding, B),with thiophene as a linker, and C),with triazole as a linker. The oligopeptides syntheses catalyzed by these magnetic nanoparticles (MNPs) with immobilized proteases were systematically studied. Our results show that i),for magnetic nanoparticles immobilized ,-chymotrypsin, both immobilization strategies A and B furnished good reusability for the Z-Tyr-Gly-Gly-OEt synthesis, the MNPs enzymes can be readily used at least five times without significant loss of its catalytic performance: ii),In the case of Z-Asp-Phe-OMe synthesis catalyzed by magnetic nanoparticles immobilized thermolysin, immobilization Strategy,B provided the best recyclability: iii),For the immobilized papain, although Strategy,A or B afforded an immobilized enzyme for the first cycle of Z-Ala-Leu-NHNHPh synthesis in good yield, their subsequent catalytic activity decreased rapidly. In general, the ,-Fe2O3 MNPs were better for use as an immobilization matrix, rather than the Fe3O4 MNPs, owing to their smaller particle size and higher surface area. [source] Enzymes in the acquired enamel pellicleEUROPEAN JOURNAL OF ORAL SCIENCES, Issue 1 2005Christian Hannig The acquired pellicle is a biofilm, free of bacteria, covering oral hard and soft tissues. It is composed of mucins, glycoproteins and proteins, among which are several enzymes. This review summarizes the present state of research on enzymes and their functions in the dental pellicle. Theoretically, all enzymes present in the oral cavity could be incorporated into the pellicle, but apparently enzymes are adsorbed selectively onto dental surfaces. There is clear evidence that enzymes are structural elements of the pellicle. Thereby they exhibit antibacterial properties but also facilitate bacterial colonization of dental hard tissues. Moreover, the immobilized enzymes are involved in modification and in homeostasis of the salivary pellicle. It has been demonstrated that amylase, lysozyme, carbonic anhydrases, glucosyltransferases and fructosyltransferase are immobilized in an active conformation in the pellicle layer formed in vivo. Other enzymes, such as peroxidase or transglutaminase, have been investigated in experimental pellicles. Despite the depicted impact of enzymes on the formation and function of pellicle, broader knowledge on their properties in the in vivo -formed pellicle is required. This might be beneficial in the development of new preventive and diagnostic strategies. [source] One-Pot Preparation of Polymer,Enzyme,Metallic Nanoparticle Composite Films for High-Performance Biosensing of Glucose and GalactoseADVANCED FUNCTIONAL MATERIALS, Issue 11 2009Yingchun Fu Abstract New polymer,enzyme,metallic nanoparticle composite films with a high-load and a high-activity of immobilized enzymes and obvious electrocatalysis/nano-enhancement effects for biosensing of glucose and galactose are designed and prepared by a one-pot chemical pre-synthesis/electropolymerization (CPSE) protocol. Dopamine (DA) as a reductant and a monomer, glucose oxidase (GOx) or galactose oxidase (GaOx) as the enzyme, and HAuCl4 or H2PtCl6 as an oxidant to trigger DA polymerization and the source of metallic nanoparticles, are mixed to yield polymeric bionanocomposites (PBNCs), which are then anchored on the electrode by electropolymerization of the remaining DA monomer. The prepared PBNC material has good biocompatibility, a highly uniform dispersion of the nanoparticles with a narrow size distribution, and high load/activity of the immobilized enzymes, as verified by transmission/scanning electron microscopy and electrochemical quartz crystal microbalance. The thus-prepared enzyme electrodes show a largely improved amperometric biosensing performance, e.g., a very high detection sensitivity (99 or 129,µA cm,2 mM,1 for glucose for Pt PBNCs on bare or platinized Au), a sub-micromolar limit of detection for glucose, and an excellent durability, in comparison with those based on conventional procedures. Also, the PBNC-based enzyme electrodes work well in the second-generation biosensing mode. The proposed one-pot CPSE protocol may be extended to the preparation of many other functionalized PBNCs for wide applications. [source] Electrospun Nanofibers Modified with Phospholipid Moieties for Enzyme ImmobilizationMACROMOLECULAR RAPID COMMUNICATIONS, Issue 16 2006Xiao-Jun Huang Abstract Summary: PANCMPCs containing phospholipid side moieties were electrospun into nanofibers with a mean diameter of 90 nm. Field emission SEM was used to characterize the morphologies of the nanofibers. These phospholipid-modified nanofibers were explored as supports for enzyme immobilization due to the characteristics of excellent biocompatibility, high surface/volume ratio, and porosity, which were beneficial to the catalytic efficiency and activity of immobilized enzymes. Lipase from Candida rugosa was immobilized on these nanofibers by adsorption. Preliminary results indicated that the properties of the immobilized lipase on these phospholipid-modified nanofibers were greatly promising. Schematic representation of the structure and electrostatic properties of phospholipid-modified nanofibers. [source] The relevance of pH gradients in immobilized enzymesBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2010Article first published online: 20 MAY 2010 No abstract is available for this article. [source] Catalytic behaviors of enzymes attached to nanoparticles: the effect of particle mobilityBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2003Hongfei Jia Abstract Nanoparticles provide an ideal remedy to the usually contradictory issues encountered in the optimization of immobilized enzymes: minimum diffusional limitation, maximum surface area per unit mass, and high effective enzyme loading. In addition to the promising performance features, the unique solution behaviors of the nanoparticles also point to a transitional region between the heterogeneous (with immobilized enzymes) and homogeneous (with soluble free enzymes) catalysis. The particle mobility, which is related to particle size and solution viscosity through Stokes-Einstein equation, may impact the reaction kinetics according to the collision theory. The mobility-activity relationship was examined through experimental studies and theoretical modeling in the present work. Polystyrene particles with diameters ranging from 110,1000 nm were prepared. A model enzyme, ,-chymotrypsin, was covalently attached to the nanoparticles up to 6.6 wt%. The collision theory model was found feasible in correlating the catalytic activities of particles to particle size and solution viscosity. Changes in the size of particles and the viscosity of reaction media, which all affect the mobility of the enzyme catalyst, evidently altered the intrinsic activity of the particle-attached enzyme. Compared to KM, kcat appeared to be less sensitive to particle size and viscosity. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng84: 406,414, 2003. [source] Controlled enzymatic removal of damaging casein layers on medieval wall paintingsBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2002Sascha Beutel Abstract A new, gentle enzymatic method was developed for a controlled removal of casein layers from medieval wall paintings. These casein layers were applied over the last 60 years on wall paintings in order to decrease substantial damage due to a peeling off of the frescoes from the roughcast surface due to environmental effects. However, due to the aging of the casein layers (at 40,50 years), a more drastic peeling occurred and the danger of total destruction of the wall paintings is severe. Thus, screening was performed to find the most suitable enzyme for casein digestion. Alcalase 2.5 DX L was the most appropriate enzyme for an effective proteolysis reaction. The enzyme was immobilized on functionalized cellulose membrane. A membrane pad system with immobilized enzymes was developed which could be pressed on the casein layers on the wall painting. A controlled removal of the casein layers by proteolytic digestion was observed and it was possible to continuously wash off the hydrolyzed casein fragments from the wall painting surface by an aqueous carbonate buffer flowing through the membrane pad. The removal and the digestion was monitored by reverse HPLC. Additionally, an on-line monitoring system was set up in order to continuously follow the casein layer removal and the digestion procedure directly on the wall painting. This technique is based on noninvasive 2D-fluorescence monitoring. Optical fiber systems were used to continuously monitor the fluorescence intensity of casein-bound tryptophan. The off-line data were verified with the on-line 2D-fluorescence data. Based on the scientific result an appropriate technique for the controlled enzymatic removal of damaging casein layers on the surface of medieval wall paintings using immobilized enzyme is now available. It is now applied to remove such casein layers from medieval wall paintings in the Allerheiligen-Kapelle Cloister, Wienhausen, Germany, and the St. Alexander Kirche, Wildeshausen, Germany. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 13,21, 2002. [source] Enzyme-Carrying Polymeric Nanofibers Prepared via Electrospinning for Use as Unique BiocatalystsBIOTECHNOLOGY PROGRESS, Issue 5 2002Hongfei Jia Improvement of catalytic efficiency of immobilized enzymes via materials engineering was demonstrated through the preparation of bioactive nanofibers. Bioactive polystyrene (PS) nanofibers with a typical diameter of 120 nm were prepared and examined for catalytic efficiency for biotransformations. The nanofibers were produced by electrospinning functionalized PS, followed by the chemical attachment of a model enzyme, ,-chymotrypsin. The observed enzyme loading as determined by active site titration was up to 1.4% (wt/wt), corresponding to over 27.4% monolayer coverage of the external surface of nanofibers. The apparent hydrolytic activity of the nanofibrous enzyme in aqueous solutions was over 65% of that of the native enzyme, indicating a high catalytic efficiency as compared to other forms of immobilized enzymes. Furthermore, nanofibrous ,-chymotrypsin exhibited a much-improved nonaqueous activity that was over 3 orders of magnitude higher than that of its native counterpart suspended in organic solvents including hexane and isooctane. It appeared that the covalent binding also improved the enzyme's stability against structural denaturation, such that the half-life of the nanofibrous enzyme in methanol was 18-fold longer than that of the native enzyme. 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