Showing papers on "Immobilized enzyme published in 2006"

Electrospun poly (vinyl alcohol)/glucose oxidase biocomposite membranes for biosensor applications

Abstract: A novel technique for enzyme immobilization on the surface of the Au electrode for designing amperometric biosensor by electrospinning poly(vinyl alcohol) (PVA) and glucose oxidase (GOD) is presented in this paper. The immobilized GOD remained active inside the electrospun PVA fibrous membranes. The membranes are promising candidates for immobilization of enzymes because of their high specific surface area and porous structure. The infrared spectrum, the UV–Vis spectrum, and the scanning electronic microscope of the membranes showed that the enzyme had been immobilized inside the PVA membranes. Chronoamperometric measurements demonstrated that electrospun fibrous enzymatic electrodes exhibited a rapid response (1 s) and a higher response current (μA level) to glucose in the normal and diabetic level. The linear response range (from 1 to 10 mM) and the lower detection limit (0.05 mM) of the sensor are satisfying. The electrospun method makes it convenient and efficient to prepare the enzymatic electrode for biosensors.

Covalent enzyme immobilization onto photopolymerized highly porous monoliths

Abstract: In recent years enzymes have found widespread use in areas as diverse as chemical synthesis, decontamination of waste streams, and biosensors. For ease of application and for stabilization purposes, enzymes are often immobilized on solid supports. These supports can include inorganic substrates such as silicon or glass, as well as organic materials such as polymers or hydrogels. In current industrial processes, enzymes are often simply adsorbed onto the material. Although this is a cost-effective method for supporting enzymes, these biocatalytic materials often suffer from decreased activity after prolonged usage because of the leakage of adsorbed enzymes during catalysis and recycling. Furthermore, since there is a lack of control over the enzyme positioning, only a fraction of the enzyme is in contact with the environment and therefore used effectively. In more sophisticated applications, such as sensors and analytical devices, covalent immobilization is achieved using carefully designed anchors and surface modification. These multistep procedures lead to welldefined systems, but costs are raised considerably and these methods are difficult to extend to large-scale applications. In this article we describe a simple and effective strategy for immobilizing enzymes covalently onto a solid porous support, and ascertain that a large fraction of the enzyme is available for catalysis. To achieve this, a support with a high surface to volume ratio is employed, namely a polymerized high internal phase emulsion (polyHIPE). N-Hydroxysuccinimide esters are introduced into this highly porous monolithic support for covalent coupling with the lysine residues present in proteins. When the activity of the well-known lipase Candida antarctica Lipase B (CAL-B) immobilized on polyHIPE was compared with the commercially available standard, Novozyme 435, a remarkable increase in both activity and stability was observed. PolyHIPEs are highly porous polymers obtained by polymerizing the continuous phase of a HIPE. The emulsion must have a droplet volume fraction of at least 0.74, and can exceed 0.99. The stability of this emulsion is mainly dependent on the nature of the surfactant, which should be soluble only in the continuous phase. PolyHIPEs have a well-defined open cellular morphology, with interconnecting holes (windows) between voids resulting from the continuous phase shrinkage during polymerization (see Fig. 1). Their unique morphology differentiates them from other porous polymers (e.g. gasblown foams), and yields excellent flow-through properties. Since the development of polyHIPEs in the 1980s, numerous applications have been developed, such as solid-phase peptide synthesis supports, catalyst supports, superabsorbents, ion-exchange resins, monolithic supports for cells, and membrane filters. However, the use of polyHIPEs as covalently immobilized enzyme supports for biocatalysis has not yet been reported. C O M M U N IC A TI O N S

Water‐soluble carbon nanotube‐enzyme conjugates as functional biocatalytic formulations

Abstract: We report the activity, stability, and reusability of enzyme-carbon nanotube conjugates in aqueous solutions. A variety of enzymes were covalently attached to oxidized multi-walled carbon nanotubes (MWNTs). These conjugates were soluble in aqueous buffer, retained a high fraction of their native activity, and were stable at higher temperatures relative to their solution phase counterparts. Furthermore, the high surface area of MWNTs afforded high enzyme loadings, yet the intrinsic high length of the MWNT led to facile filtration. These water-soluble carbon nanotube-enzyme conjugates represent novel preparations that possess the virtues of both soluble and immobilized enzymes, thus providing a unique combination of useful attributes such as low mass transfer resistance, high activity and stability, and reusability.

Porous Gold-Nanoparticle−CaCO3 Hybrid Material: Preparation, Characterization, and Application for Horseradish Peroxidase Assembly and Direct Electrochemistry

Abstract: Gold nanoparticles (AuNPs) were assembled on the surface of porous calcium carbonate microspheres (CaCO3) in a neutral aqueous solution through electrostatic interaction. The resulting gold-nanoparticle−CaCO3 hybrid material (AuNP−CaCO3) was characterized and expected to offer a promising template for enzyme immobilization and biosensor fabrication because of its satisfying biocompatibility and improved properties. It was conjugated with horseradish peroxidase (HRP) to fabricate HRP−AuNP−CaCO3 bioconjugates, which were then embedded into a silica sol−gel matrix to construct a novel biosensor. Because of the synergic effect of CaCO3 microspheres and AuNPs, direct electron transfer of HRP was observed at this biosensor. The biosensor exhibited a fast amperometric response to H2O2, with a linear range of 4.0 × 10-5 to 8.0 × 10-3 mol/L. The detection limit was 1.0 × 10-6 mol/L based on S/N = 3.

Less common applications of monoliths: I. Microscale protein mapping with proteolytic enzymes immobilized on monolithic supports.

Abstract: This review summarizes the recent contributions to the rapidly growing area of immobilized enzymes employing both silica and synthetic polymer-based monoliths as supports. Focus is mainly on immobilized proteolytic enzyme reactors designed for studies in proteomics. Porous monoliths emerged first as a new class of stationary phases for HPLC in the early 1990s. Soon thereafter, they were also used as supports for immobilization of proteins and preparation of both stationary phases for bioaffinity chromatography and enzymatic reactors. Organic polymer-based monoliths are typically prepared using a simple molding process carried out within the confines of a "mold" such as chromatographic column or capillary. Polymerization of a mixture comprising monomers, initiator, and porogenic solvent affords macroporous materials. In contrast, silica-based monoliths are first formed as a rigid rod from tetraalkoxysilane in the presence of PEG and subsequently encased with a plastic tube. Both types of monolith feature large through-pores that enable a rapid flow-through. Since all the solutions must flow through the monolith, the convection considerably accelerates mass transfer within the monolith. As a result, reactors including enzyme immobilized on monolithic support exhibit much higher activity compared to the reactions in solution.

Immobilization of beta-glucosidase on Eupergit C for lignocellulose hydrolysis

Abstract: beta-Glucosidase is frequently used to supplement cellulase preparations for hydrolysis of cellulosic and lignocellulosic substrates in order to accelerate the conversion of cellobiose to glucose. Typically, commercial cellulase preparations are deficient in this enzyme and accumulation of cellobiose leads to product inhibition. This study evaluates the potential for recycling beta-glucosidase by immobilization on a methacrylamide polymer carrier, Eupergit C. The immobilized beta-glucosidase had improved stability at 65 degrees C, relative to the free enzyme, while the profile of activity versus pH was unchanged. Immobilization resulted in an increase in the apparent Km from 1.1 to 11 mM: and an increase in Vmax from 296 to 2430 micromol mg(-1) min(-1). The effect of immobilized beta-glucosidase on the hydrolysis of cellulosic and lignocellulosic substrates was comparable to that of the free enzyme when used at the same level of protein. Operational stability of the immobilized beta-glucosidase was demonstrated during six rounds of lignocellulose hydrolysis.

Multilayer-Assembled Microchip for Enzyme Immobilization as Reactor Toward Low-Level Protein Identification

Abstract: A microchip reactor has been developed on the basis of a layer-by-layer approach for fast and sensitive digestion of proteins. The resulting peptide analysis has been carried out by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Natural polysaccharides, positively charged chitosan (CS), and negatively charged hyaluronic acid (HA) were multilayer-assembled onto the surface of a poly(ethylene terephthalate) (PET) microfluidic chip to form a microstructured and biocompatible network for enzyme immobilization. The construction of CS/HA assembled multilayers on the PET substrate was characterized by AFM imaging, ATR-IR, and contact angle measurements. The controlled adsorption of trypsin in the multilayer membrane was monitored using a quartz crystal microbalance and an enzymatic activity assay. The maximum proteolytic velocity of the adsorbed trypsin was ∼600 mM/min μg, thousands of times faster than that in solution. BSA, myoglobin, and cytochrome c were used as ...

Immobilization of lipase from Candida rugosa on electrospun polysulfone nanofibrous membranes by adsorption

Abstract: Polysulfone composite nanofibrous membranes were prepared by electrospinning and were used to immobilize lipase from Candida rugosa by physical adsorption. Field emission scanning electron microscopy was used to evaluate the morphology and diameter of the nanofibers. PVP and PEG were used as additives to render the nanofibrous membranes biocompatibility favored by immobilized enzyme. Effects of post-treatment, additive concentration, pH and temperature were investigated on the adsorption capacity and activity of immobilization preparations, as well as thermal stability. It was found that (1) post-treatment had no significant effect on the adsorption capacity; (2) the increment of PVP or PEG concentration was negative for the adsorption capacity but positive for activity of immobilized lipase; (3) the immobilized lipase showed less sensitivity for pH and higher optimum temperature. Thermal stability for the immobilization preparations was enhanced compared with that for free preparations. The kinetic parameters of the free and immobilized lipases, Km and Vmax were also assayed. Results indicated that Km and Vmax for the immobilized lipases were higher and lower than those for free lipase, respectively.

Immobilization of lipases for non-aqueous synthesis

Abstract: Immobilization of lipases involves many levels of complications relating to the structure of the active site and its interactions with the immobilization support. Interaction of the so called hydrophobic ‘lid’ with the support has been reported to affect synthetic activity of an immobilized lipase. In this work we evaluate and compare the synthetic activity of lipases from different sources immobilized on different kinds of supports with varying hydrophobicity. Humicola lanuginosa lipase, Candida antarctica lipase B and Rhizomucor miehei lipase were physically adsorbed onto two types of hydrophobic carriers, namely hydrophilic carriers with conjugated hydrophobic ligands, and supports with base matrix hydrophobicity. The prepared immobilized enzymes were used for acylation of n -butanol with oleic acid as acyl donor in iso-octane with variable water content (0–2.8%, v/v) as reaction medium. Enzyme activity and effect of water on the activity of the immobilized derivatives were compared with those of respective soluble lipases and a commercial immobilized lipase Novozyme 435. Both R. miehei and H. lanuginosa immobilized lipases showed maximum activity at 1.39% (v/v) added water concentration. Sepabeads, a methacrylate based hydrophilic support with conjugated octadecyl chain showed highest immobilized esterification (synthetic) activity for all three enzymes, and of the three R. miehei lipase displayed maximum esterification activity comparable to the commercial enzyme.

Surface Functionalized Electrospun Biodegradable Nanofibers for Immobilization of Bioactive Molecules

Abstract: A blend mixture of biodegradable poly(epsilon-caprolactone) (PCL) and poly(d,l-lactic-co-glycolic acid)-poly(ethylene glycol)-NH(2) (PLGA-b-PEG-NH(2)) block copolymer was electrospun to produce surface functionalized nanofibers. The resulting nanofibrous mesh with primary amine groups on the surface was applied for immobilization of biologically active molecules using lysozyme as a model enzyme. Lysozyme was immobilized via covalent conjugation by using a homobifunctional coupling agent. The nanofibrous mesh could immobilize a far greater amount of lysozyme on the surface with concomitantly increased activity, primarily due to its larger surface area, compared to that of the solvent casting film. It was also found that the enzyme immobilization process slightly altered thermal and pH-dependent catalytic activity profiles compared to those of native lysozyme. The results demonstrated the surface functionalized electrospun nanofibrous mesh could be used as a promising material for immobilizing a wide range of bioactive molecules.

Characterization of functionalized nanoporous supports for protein confinement

Abstract: Here we characterize a highly efficient approach for protein confinement and enzyme immobilization in NH2– or HOOC– functionalized mesoporous silica (FMS) with pore sizes as large as tens of nanometres. We observed a dramatic increase of enzyme loading in both enzyme activity and protein amount when using appropriate FMS in comparison with unfunctionalized mesoporous silica and normal porous silica. With different protein loading density in NH2–FMS, the negatively charged glucose oxidase (GOX) displayed an immobilization efficiency (Ie, the ratio of the specific activity of the immobilized enzyme to the specific activity of the free enzyme in stock solution) in a range from 30% to 160%, while the same charged glucose isomerase (GI) showed an Ie of 100% to 120%, and the positively charged organophosphorus hydrolase (OPH) exhibited Ie of more than 200% in HOOC–FMS. The enzyme–FMS composite was stained with the charged gold nanoparticles and imaged by transmission electron microscopy (TEM). Fourier transform infrared (FTIR) spectroscopy showed no major secondary structural change for the enzymes entrapped in FMS. Thanks to the large, rigid, open pore structure of FMS, the reaction rate and Km of the entrapped enzymes in FMS were comparable to those of the free enzymes in solution. In principle, the general approach described here should be applicable to many enzymes, proteins, and protein complexes since both pore sizes and functional groups of FMS are controllable.

Chemical modification of papain for use in alkaline medium

Abstract: Chemical modification is a useful method to recognize and modify functional determinants of enzymes. Papain, an endolytic cysteine protease (EC3.4.22.2) from Carica papaya latex has been chemically modified using different dicarboxylic anhydrides of citraconic, phthalic, maleic and succinic acids. These anhydrides reacted with five to six amino groups of the lysine residues in the enzyme, thereby changing the net charge of the enzyme from positive to negative. The resultant enzyme had its optimum pH shifted from 7 to 9 and change in temperature optima from 60 to 80 °C. The modified papain also had a higher thermostability. Stability of the modified papain was further increased by immobilization of the enzyme either by adsorption onto inert matrix or by entrapment in polysaccharide polymeric gels. Entrapment in starch gel showed better retention of enzyme activity. Incorporation of modified and immobilized enzymes to branded domestic detergent powders was found to have very good activity retention. The papain entrapped in starch gel showed better stability and activity retention than in other carbohydrate polymers when added to domestic detergent powders.