Showing papers on "Immobilized enzyme published in 2002"

Smart nanotubes for bioseparations and biocatalysis.

Abstract: Tube-shaped nanostructures (nanotubes) have a number of attributes that make them potentially useful for biomedical applications such as drug delivery/detoxification and enzyme immobilization. Template synthesis provides a route for preparing monodisperse nanotubes of nearly any size and composed of nearly any material. We show here that template-synthesized silica nanotubes can be biochemically functionalized such that they act as biocatalysts and highly selective nano-phase extraction agents for bioseparations. For example, nanotubes containing an enantioselective antibody selectively extract the enantiomer of a drug molecule that binds to the antibody, relative to the enantiomer that has no specific interaction with the antibody. Nanotubes containing the enzyme glucose oxidase function as nanophase bioreactors to catalyze the oxidation of glucose.

Entrapping enzyme in a functionalized nanoporous support.

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

Enzyme-Carrying Polymeric Nanofibers Prepared via Electrospinning for Use as Unique Biocatalysts

Abstract: 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, alpha-chymotrypsin The observed enzyme loading as determined by active site titration was up to 14% (wt/wt), corresponding to over 274% 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 alpha-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

Enzyme Kinetics: A Modern Approach

Abstract: Preface. Tools and Techniques of Kinetic Analysis. How do Enzymes Work? Characterization of Enzyme Activity. Reversible Enzyme Inhibition. Irreversible Enzyme Inhibition. pH Dependence of Enzyme-Catalyzed Reactions. Two-Substrate Reactions. Multisite and Cooperative Enzymes. Immobilized Enzymes. Interfacial Enzymes. Transient Phases of Enzymatic Reactions. Characterization of Enzyme Stability. Mechanism-Based Inhibition (Leslie J. Copp). Putting Kinetic Principles into Practice (Kirk L. Parkin). Use of Enzyme Kinetic Data in the Study of Structure-Function Relationships of Proteins (Takuji Tanaka and Rickey Y. Yada). Bibliography. Index.

Epoxy sepabeads: a novel epoxy support for stabilization of industrial enzymes via very intense multipoint covalent attachment.

Abstract: Sepabeads-EP (a new epoxy support) has been utilized to immobilize-stabilize the enzyme penicillin G acylase (PGA) via multipoint covalent attachment. These supports are very robust and suitable for industrial purposes. Also, the internal geometry of the support is composed by cylindrical pores surrounded by the convex surfaces (this offers a good geometrical congruence for reaction with the enzyme), and it has a very high superficial density of epoxy groups (around 100 micromol/mL). These features should permit a very intense enzyme-support interaction. However, the final stability of the immobilized enzyme is strictly dependent on the immobilization protocol. By using conventional immobilization protocols (neutral pH values, nonblockage of the support) the stability of the immobilized enzyme was quite similar to that achieved using Eupergit C to immobilize the PGA. However, when using a more sophisticated three-step immobilization/stabilization/blockage procedure, the Sepabeads derivative was hundreds-fold more stable than Eupergit C derivatives. The protocol used was as follows: (i) the enzyme was first covalently immobilized under very mild experimental conditions (e.g., pH 7.0 and 20 degrees C); (ii) the already immobilized enzyme was further incubated under more drastic conditions (higher pH values, long incubation periods, etc.) in order to "facilitate" the formation of new covalent linkages between the immobilized enzyme molecule and the support; (iii) the remaining epoxy groups of the support were blocked with very hydrophilic compounds to stop any additional interaction between the enzyme and the support. This third point was found to be critical for obtaining very stable enzymes: derivatives blocked with mercaptoethanol were much less stable than derivatives blocked with glycine or other amino acids. This was attributed to the better masking of the hydrophobicity of the support by the amino acids (having two charges).

Cross-linked enzyme aggregates with enhanced activity: application to lipases

Abstract: Seven commercially available microbial lipases were immobilised as their cross-linked enzyme aggregates (CLEAs). Preparations with enhanced activity were obtained by a judicious choice of the precipitant [(NH4)2SO4, 1,2-dimethoxyethane or acetone] and by adding either a crown ether or surfactant, depending on the source of the enzyme. Thus, precipitation of the lipases from Thermomyces lanuginosus and Rhizomucor miehei with (NH4)2SO4 in the presence of SDS, followed by cross-linking with glutaraldehyde, afforded CLEAs with three and two times, respectively, the hydrolytic activity of the native enzymes. Preparations with up to ten times enhanced activity in organic medium were similarly prepared.

Enzyme Based Amperometric Biosensors for Food Analysis

Abstract: This review introduces the principles of amperometric detection e.g. oxygen electrodes, hydrogen peroxide electrodes, NADH detection, mediators-aid detection, conductive organic salts and wiring electrodes. A short categorization and description of the materials commonly used for the construction of electrodes, e.g., platinum, glassy carbon, different types of graphite, screen-printed electrodes, rigid carbon-polymer biocomposites, zeolites, clays, and polymeric membranes is given. Approaches to construction of biosensors with respect to various strategies of enzyme immobilization, e.g., physical binding, covalent binding, gel entrapment, electropolymerization, sol-gel techniques and self-assembled architectures are also presented. The requirements and problems for sensing in food industry, examples of enzyme electrodes, published in the literature during the last half-decade, commercial biosensors released into the market along with the current and modern instrumentation, are also presented.

Catalytic activity of mesoporous silicate-immobilized chloroperoxidase

Abstract: A versatile enzyme, FeHeme chloroperoxidase (CPO) from Caldariomyces fumago, is immobilized in the mesoporous silicate material, mesocellular foam (MCF). MCF is a promising material for immobilizing enzymes, due to its large pore structure and high loading capacity compared to other mesoporous materials, such as MCM-48, SBA-16 and SBA-15. The immobilized CPO in MCF retains its activity. The optimal pH at which the maximum amount of enzyme is immobilized was determined to be pH 3.4, slightly below the isoelectric point of the enzyme. A weak ionic interaction between the enzyme and the surface of the inorganic substrate is thought to be critical in maintaining the activity of the immobilized enzyme. The loading capacity of MCF is 122 mg protein per 1 g of MCF. We demonstrate the advantage of MCF as an inorganic substrate for immobilization of enzymes.

Design and characterization of immobilized enzymes in microfluidic systems.

Abstract: Herein we report the fabrication, characterization, and use of total analytical microsystems containing surface-immobilized enzymes. Streptavidin-conjugated alkaline phosphatase was linked to biotinylated phospholipid bilayers coated inside poly(dimethylsiloxane) microchannels and borosilicate microcapillary tubes. Rapid determination of enzyme kinetics at many different substrate concentrations was made possible by carrying out laminar flow-controlled dilution on-chip. This allowed Lineweaver−Burk analysis to be performed from a single experiment with all the data collected simultaneously. The results revealed an enzyme turnover number of 51.1 ± 3.2 s-1 for this heterogeneous system. Furthermore, the same enzyme immobilization strategy was extended to demonstrate that multiple chemical reactions could be performed in sequence by immobilizing various enzymes in series. Specifically, the presence of glucose was detected by two coupled steps employing immobilized avidinD-conjugated glucose oxidase and strep...

Production of galacto-oligosaccharides from lactose by Aspergillus oryzae beta-galactosidase immobilized on cotton cloth.

Abstract: The production of galacto-oligosaccharides (GOS) from lactose by A. oryzae beta-galactosidase immobilized on cotton cloth was studied. The total amounts and types of GOS produced were mainly affected by the initial lactose concentration in the reaction media. In general, more and larger GOS can be produced with higher initial lactose concentrations. A maximum GOS production of 27% (w/w) of initial lactose was achieved at 50% lactose conversion with 500 g/L of initial lactose concentration. Tri-saccharides were the major types of GOS formed, accounting for more than 70% of the total GOS produced in the reactions. Temperature and pH affected the reaction rate, but did not result in any changes in GOS formation. The presence of galactose and glucose at the concentrations encountered near maximum GOS greatly inhibited the reactions and reduced GOS yield by as much as 15%. The cotton cloth as the support matrix for enzyme immobilization did not affect the GOS formation characteristics of the enzyme, suggesting no diffusion limitation in the enzyme carrier. The thermal stability of the enzyme increased approximately 25-fold upon immobilization on cotton cloth. The half-life for the immobilized enzyme on cotton cloth was more than 1 year at 40 degrees C and 48 days at 50 degrees C. Stable, continuous operation in a plugflow reactor was demonstrated for 2 weeks without any apparent problem. A maximum GOS production of 21 and 26% (w/w) of total sugars was attained with a feed solution containing 200 and 400 g/L of lactose, respectively, at pH 4.5 and 40 degrees C. The corresponding reactor productivities were 80 and 106 g/L/h, respectively, which are at least several-fold higher than those previously reported.

Hydrolysis of oils by using immobilized lipase enzyme: A review

Abstract: This review focuses on the use of immobilized lipase technology for the hydrolysis of oils. The importance of lipase catalyzed fat splitting process, the various immobilization procedures, kinetics, deactivation kinetics, New immobilized lipases for chiral resolution, reactor configurations, and process considerations are all reviewed and discussed.

Immobilized lipase‐catalysed production of alkyl esters of restaurant grease as biodiesel

Abstract: Simple alkyl ester derivatives of restaurant grease were prepared using immobilized lipases as biocatalysts. The lipases studied included those of Thermomyces lanuginosa and Candida antarctica supported on granulated silica (gran- T.l. and gran- C.a., respectively), C. antarctica supported on a macroporous acrylic resin (SP435) and Pseudomonas cepacia immobilized within a phyllosilicate sol-gel matrix (IM PS-30). All alcoholysis reactions were carried out in solvent-free media employing a one-step addition of the alcohol to the reaction system. Of the lipases studied, IM PS-30 was found to be the most effective in catalysing the methanolysis and ethanolysis of grease. The processes catalysed by gran- T.l. and gran- C.a. lipases gave poor conversions to esters, and the SP435-catalysed reactions gave intermediate yields of ethyl and methyl esters. Water activity (a(w)) was an important factor in the methanolysis reactions; reaction media with a(w)<0.5 resulted in the highest conversions to methyl esters. Molecular sieves also improved methyl ester yields by as much as 20% in transesterification reactions catalysed by IM PS-30. The immobilized lipases also were evaluated for their ability to produce alkyl esters of grease with several additional normal and branched-chain alcohols.

Degradation of textile dyes mediated by plant peroxidases.

Abstract: The peroxidase enzyme from the plants Ipomea palmata (1.003 IU/g of leaf) and Saccharum spontaneum (3.6 IU/g of leaf) can be used as an alternative to the commercial source of horseradish and soybean peroxidase enzyme for the decolorization of textile dyes, mainly azo dyes. Eight textiles dyes currently used by the industry and seven other dyes were selected for decolorization studies at 25-200 mg/L levels using these plant enzymes. The enzymes were purified prior to use by ammonium sulfate precipitation, and ion exchange and gel permeation chromatographic techniques. Peroxidase of S. spontaneum leaf (specific activity of 0.23 IU/mg) could completely degrade Supranol Green and Procion Green HE-4BD (100%) dyes within 1 h, whereas Direct Blue, Procion Brilliant Blue H-7G and Chrysoidine were degraded >70% in 1 h. Peroxidase of Ipomea (I. palmata leaf; specific activity of 0.827 U/mg) degraded 50 mg/L of the dyes Methyl Orange (26%), Crystal Violet (36%), and Supranol Green (68%) in 2-4 h and Brilliant Green (54%), Direct Blue (15%), and Chrysoidine (44%) at the 25 mg/L level in 1 to 2 h of treatment. The Saccharum peroxidase was immobilized on a hydrophobic matrix. Four textile dyes, Procion Navy Blue HER, Procion Brilliant Blue H-7G, Procion Green HE-4BD, and Supranol Green, at an initial concentration of 50 mg/L were completely degraded within 8 h by the enzyme immobilized on the modified polyethylene matrix. The immobilized enzyme was used in a batch reactor for the degradation of Procion Green HE-4BD and the reusability was studied for 15 cycles, and the half-life was found to be 60 h.

Modulation of the enantioselectivity of Candida antarctica B lipase via conformational engineering. Kinetic resolution of (±)-α-hydroxy-phenylacetic acid derivatives

Abstract: The modulation, via immobilization and engineering the reaction medium, of the enantioselectivity exhibited by the lipase from Candida antarctica B (CABL) in the hydrolysis of α-hydroxy-phenylacetic acid derivatives is shown. The enzyme was purified and immobilized using different protocols to obtain immobilized enzyme preparations with different orientations and micro-environments. The catalytic properties (activity, specificity, enantioselectivity) of the resulting derivatives were found to be quite different from each other. The enantioselectivity ( E value) strongly depends on the type of derivative and the conditions employed. Thus, the enzyme immobilized on cyanogen bromide (CNBr) presented E =7.4, while the PEI derivative yielded E =67 in the hydrolysis of α-hydroxy-phenylacetic acid methyl ester under similar conditions. Moreover, the enantioselectivity of the PEI derivative decreased from 67 to 14 on lowering the reaction temperature from 25 to 4°C at pH 5, while the E of some other derivatives improved significantly under similar experimental changes. Similar changes in the E values were observed in the hydrolysis of ( RS )-2-butyroyl-2-phenylacetic acid. Using this substrate, the interfacially adsorbed enzyme (octadecyl) afforded an E value of only 2 at pH 5, while the glutaraldehyde derivative presented a high enantioselectivity ( E >400) under all conditions studied. The corresponding ( S )-ester and ( R )-acid were obtained with excellent enantiomeric excess using the glutaraldehyde derivative, while using the interfacially immobilized one there was no appreciable enantioselectivity. Thus, using differently immobilized derivatives and different experimental conditions, lipase enantioselectivity could vary from negligible to up to 400. The experimental conditions were also found to have varying effects on the different lipase derivatives.

Oxidation of ABTS by hydrogen peroxide catalyzed by horseradish peroxidase encapsulated into sol–gel glass.: Effects of glass matrix on reactivity

Abstract: Encapsulation of horseradish peroxidase (HRP) by the sol–gel method into silica (SiO 2 ) or alkylated silica (RSiO 2 , in which R is Me or Pr) yields biocatalytic glasses designated HRP@SiO 2 and HRP@RSiO 2 . These new enzyme composite materials catalyze one-electron oxidation by H 2 O 2 of the dye 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) into the radical cation ABTS + . In the presence of excess H 2 O 2 , ABTS + is converted into a mixture of at least five compounds. This undesirable side-reaction can be suppressed by using stoichiometric amount of H 2 O 2 . The effects of ABTS concentration and ionic strength of the buffer on the ABTS + yield and the apparent rate constant were investigated. The catalyst HRP@MeSiO 2 achieves higher ABTS + yields (70–85%) than HRP@SiO 2 and HRP@PrSiO 2 (30–40%) do. The apparent rate constants for HRP@MeSiO 2 are 7–10 times higher than those for HRP@SiO 2 and HRP@PrSiO 2 . When the ionic strength of the buffer is raised, the rate constants increase for HRP@SiO 2 and HRP@PrSiO 2 , and do not change for HRP@MeSiO 2 . The diminished electrostatic interaction between the negatively-charged substrate, ABTS, and the surface of methylated silica is the main cause for the increased catalytic efficiency. These results are important for understanding of encapsulated and otherwise immobilized enzymes. The possible electrostatic effects should be taken into consideration in the choice of the matrix materials used in the design of biosensors, supported catalysts, and other composite materials.

Observation of topical catalysis by sphingomyelinase coupled to microspheres.

Abstract: Sphingomyelinase, SMase (EC 3.1.4.12), was coupled onto amino-derivatized acrylate microspheres and was shown to retain its catalytic activity. The immobilized enzyme allows one to carry out topical enzymatic reaction in a controlled manner. Accordingly, these spheres were held with a micropipet and using micromanipulator brought into contact with a giant liposome membrane composed of phosphatidylcholine and sphingomyelin (SOPC/C16:0-SM, 0.75:0.25, molar ratio), representing the substrate for the immobilized enzyme. The macroscopic consequences of the enzyme reaction were visualized using fluorescence microscopy as well as differential interference contrast microscopy. The surface contact of the giant vesicle and immobilized enzyme causes membrane microdomain formation and domain clustering (capping) in the membrane and subsequent shedding of small vesicles from the membrane into the interior of the giant liposome. The method described represents a novel approach to study enzymatic reactions and allows manipulating giant vesicles as well as cultured cells in a spatially controlled manner.

Amperometric TNT biosensor based on the oriented immobilization of a nitroreductase maltose binding protein fusion.

Abstract: The preparation and characterization of an amperometric 2,4,6-trinitrotoluene (TNT) biosensor based on the surface immobilization of a maltose binding protein (MBP) nitroreductase (NR) fusion (MBP−NR) onto an electrode modified with an electropolymerized film of N-(3-pyrrol-1-ylpropyl)-4,4‘-bipyridine (PPB) are described. The MBP domain of MBP−NR exhibits a high and specific affinity toward electropolymerized films of PPB with the immobilized enzyme retaining virtually all of its enzymatic activity. Under similar conditions, the wild-type NR enzyme (i.e., without the MBP domain) loses most of its enzymatic activity. The kinetics of the catalytic reaction between the biosensor and TNT and 2,4-dinitrotoluene (DNT) were characterized using rotated disk electrode and cyclic voltammetry techniques, and values of 1.4 × 104 and 7.1 × 104 M-1 s-1 were obtained for TNT and DNT, respectively. The apparent Michaelis−Menten constants (KM) for MBP−NR in solution and on the surface, using TNT as substrate, were determi...