Covalent attachment of cholesterol oxidase and horseradish peroxidase on perlite through silanization: activity, stability and co-immobilization

In the present work, co-immobilization of cholesterol oxidase (COD) and horseradish peroxidase (POD) on perlite surface was attempted. The surface of perlite were activated by 3-aminopropyltriethoxysilane and covalently bonded with COD and POD via glutaraldehyde. Enzymes activities have been assayed by spectrophotometric technique. The stabilities of immobilized COD and POD to pH were higher than those of soluble enzymes and immobilization shifted optimum pH of enzymes to the lower pH. Heat inactivation studies showed improved thermostability of the immobilized COD for more than two times, but immobilized POD was less thermostable than soluble POD. Also activity recovery of immobilized COD was about 50% since for immobilized POD was 11%. The K(m) of immobilized enzymes was found slightly lower than that of soluble enzymes. Immobilized COD showed inhibition in its activity at high cholesterol concentration which was not reported for soluble COD before. Co-immobilized enzymes retained 65% of its initial activity after 20 consecutive reactor batch cycles.     

Immobilized enzyme system for determination of sialic acid in serum or urine.

An immobilized enzyme system has been developed and employed to determine the concentration of sialic acid (N-acetylneuraminic acid) in human serum and urine. Two enzyme pairs, neuramindiase-Neu-5-Ac lyase and pyruvate oxidase-peroxidase, have been respectively co-immobilized onto 1,12-aminododecane-agarose with glutaraldehyde. The relative specific activity of the co-immobilized neuraminidase and Neu-5-Ac lyase were 60% and 78%, and those of pyruvate oxidase and peroxidase were 50% and 95% of the corresponding soluble enzymes, respectively. The optimal reaction pH at 37 degrees C for each of the co-immobilized enzymes was about one pH unit higher than that of the corresponding soluble enzyme. The optimal reaction temperature of each enzyme was increased as a result of immobilization. The thermal stability at 45 degrees C of the immobilized neuraminidase, Neu-5-Ac lyase, pyruvate oxidase, and peroxidase were increased 80-, 83-, 115-, and 147-fold, respectively. Km and Vm of each immobilized and co-immobilized enzyme have also been determined. The system provided a convenient and rapid method to determine the concentration of total sialic acid without pretreatment of the sample. The results correlated satisfactorily with those obtained by using a soluble enzyme system. The co-immobilized enzymes were stable for at least 1 year of 500 tests when used repeatedly. The system is thus a reproducible and reliable novel assay method for sialic acid in the serum or urine sample.     

Sol-gel encapsulated enzyme arrays for high-throughput screening of biocatalytic activity

We developed versatile low-cost arrays of sol-gel-encapsulated enzymes (referred to as solzymes) suitable for repeated assays of bioactivity or enzyme inhibition. Sol-gel microstructures containing active enzymes were stabilized on glass at moderate pH and room temperature without harsh calcination. A multi-well bilayer of polydimethylsiloxane was used to support the solzyme array and contain the reaction medium. Each of the 147 microwells has a working volume of 5 muL and contains 50 mug of immobilized enzyme. The solzyme arrays maintained high activity through repeated applications and exhibited superior thermostability compared to soluble enzymes. Among the enzymes used were lipases, glucose oxidase, and horseradish peroxidase. Twenty different lipases and proteases were also used to prepare a hydrolase array, for which bromthymol blue served as a generic indicator of activity. The relative activities of the encapsulated hydrolases correlated closely with those of the soluble hydrolases, illustrating that sol-gel encapsulation preserved the hierarchy of enzyme activity. The development of solzyme arrays paves the way to higher throughput screening of diverse proteins and enzymes, including those that are available only in trace amounts.     

Properties of enzymes immobilized by the diazotized m-diaminobenzene method.

Some properties of a number of enzymes immobilized by the diazotized m-diaminobenzene (dDAB) method are described. The pH-activity profiles of beta-D-glucosidase, glucoamylase, peroxidase, uricase, and D-glucose oxidase were virtually unchanged on immobilization while those of catalase and dextranase were significantly altered. beta-D-Glucosidase, glucoamylase, and glucose oxidase were found to be more susceptible to denaturation on lyophilization when immobilized than in the native state; however, sorbitol had a marked protective effect in every case examined. Sorbitol was also found to exert a stabilizing effect when lyophilized immobilized preparations were stored. Immobilization marginally improved the stabilities of a number of enzymes to heating at 60 degrees at pH 8.0. The usefulness for continuous reaction of a column of glucoamylase attached to celite was established. The reuse of the solid supports was demonstrated.     

Immunoaffinity layering of enzymes

A general procedure for the high yield immobilization of enzymes with the help of specific anti-enzyme antibodies is described. Polyclonal antibodies were raised against Aspergillus niger glucose oxidase and horseradish peroxidase in rabbits and the gamma globulin (IgG) fraction from the immune sera isolated by ammonium sulphate fractionation followed by ion-exchange chromatography. Immobilization of glucose oxidase and horseradish peroxidase was achieved by initially binding the enzymes to a Sepharose matrix coupled with IgG isolated from anti-(glucose oxidase) and anti-(horseradish peroxidase) sera, respectively. This was followed by alternate incubation with the IgG and the enzyme to assemble layers of enzyme and antibody on the support. The immunoaffinity-layered preparations obtained thus were highly active and, after six binding cycles, the amount of enzyme immobilized could be raised about 25 times over that bound initially. It was also possible to assemble layers of glucose oxidase using unfractionated antiserum in place of the IgG. The bioaffinity-layered preparations of glucose oxidase and horseradish peroxidase exhibited good enzyme activities and improved resistance to heat-induced inactivation. The sensitivity of a flow injection analysis system for measuring glucose and hydrogen peroxide could be remarkably improved using immunoaffinity-layered glucose oxidase and horseradish peroxidase. For the detection of glucose, a Clark-type oxygen electrode, constructed as a small flow-through cell integrated with a cartridge bearing immunoaffinity-layered glucose oxidase was employed. The hydrogen peroxide concentration was analysed spectrophotometrically using a flow-through cell and the layered horseradish peroxidase packed into a cartridge. The immunoaffinity-layered enzymes could be conveniently solubilized at acid pH and fresh enzyme loaded onto the support. Immunoaffinity-layered glucose oxidase was successfully used for the on-line monitoring of the glucose concentration during the cultivation of Streptomyces cerevisiae. Received: 16 November 1998 / Received revision: 22 March 1999 / Accepted: 26 March 1999     

Effects of immobilization on the kinetics of enzyme-catalyzed reactions. I. Glucose oxidase in a recirculation reactor system.

Glucose oxidase from Aspergillus niger was immobilized on nonporous glass beads by covalent bonding and its kinetics were studied in a packed-column recycle reactor. The optimum pH of the immobilized enzyme was the same as that of soluble enzyme; however, immobilized glucose oxidase showed a sharper pH-activity profile than that of the soluble enzyme. The kinetic behavior of immobilized glucose oxidase at optimum pH and 25 degrees C was similar to that of the soluble enzyme, but the immobilized material showed increased temperature sensitivity. Immobilized glucose oxidase showed no loss in activity on storage at 4 degrees C for nearly ten weeks. On continuous use for 60 hr, the immobilized enzyme showed about a 40% loss in activity but no change in the kinetic constant.     

Immobilization of glycoenzymes through carbohydrate side chains.

Glucoamylase, peroxidase, glucose oxidase, and carboxypeptidase Y were covalently bound to water-insoluble supports through their carbohydrate side chains. Two approaches were used. First, the carbohydrate portions of the enzymes were oxidized with periodate to generate aldehyde groups. Treatment with amines (ethylenediamine or glycyltyrosine) and borohydride provided groups through which the protein could be immobilized. Ethylenediamine was attached to glucoamylase, peroxidase, glucose oxidase, and carboxypeptidase Y to the extent of 24, 20, 30, and 15 mol/mol of enzyme, respectively. These derivatives were coupled to an aminocaproate adduct of CL-Sepharose via an N-hydroxysuccinimide ester or to CNBr-activated Sepharose. Coupling yields were in the range of 37–50%. Retained activities of the bound aminoalkyl-enzymes were 41% (glucoamylase), 79% (peroxidase), 71% (glucose oxidase), 83% (carboxypeptidase Y). A glycyltyrosine derivative of carboxypeptidase Y was bound to diazotized arylamine-glass. Coupling yield was 42% and retained esterase activity was 84%. In the second approach, the enzyme was adsorbed to immobilized concanavalin A and the complex was crosslinked. Adsorption of carboxypeptidase Y on immobilized concanavalin A followed by crosslinking with glutaraldehyde was also effective. The bound enzyme retained 96% of the native esterase activity and showed very good operational stability.     

Introduction of functional groups onto polypropylene and polyethylene surfaces for immobilization of enzymes

Polypropylene and polyethylene surfaces are activated by introducing an active functional group through 1-fluoro-2 nitro-4-azidobenzene by UV irradiation. Horseradish peroxidase and glucose oxidase are immobilized onto the activated surfaces, simply by incubating the enzymes at 37 degrees C. When untreated surfaces are used, insignificant immobilization of the enzymes is observed.     

Amperometric determination of glucose,based on the direct electron transfer between glucose oxidase and tin oxide

An amperometric glucose biosensor was designed for the detection of glucose in blood, urine, beverages, and fermentation systems. In typical glucose biosensors that employ enzymes, mediators are used for efficient electron transfer between the enzymes and the electrode. However, some of these mediators are known to be toxic to the enzymes and also must be immobilized on the surface of the electrode. We propose a mediator-free glucose biosensor that uses a glucose oxidase immobilized on a tin oxide electrode. Direct electron transfer is possible in this system because the tin oxide has redox properties similar to those of mediators. The method for immobilization of the glucose oxidase onto the tin oxide is also very simple. Tin oxide was prepared by the anodization and annealing of pure tin, and this provides a large surface area for the immobilization step because of its porosity. Glucose oxidase was immobilized onto the tin oxide using the membrane entrapment method. The proposed method provides a simple process for fabricating the enzyme electrode. Glucose oxidase immobilized onto the tin oxide, prepared in accordance with this method, has a relatively large current response when comparedto those of other glucose biosensors. The sensitivity of the biosensor was 19.55 μA/mM, and a linear response was observed between 0∼3 mM glucose. This biosensor demonstrated good reproducibility and stability.     

Preparation and characterization of alkaline phosphatase,horseradish peroxidase,and glucose oxidase conjugates with carboxylated carbon nano-onions

Carbon nanomaterials have emerged as suitable supports for enzyme immobilization and stabilization due to their inherently large surface area, high electrical conductivity, chemical stability, and mechanical strength. In this paper, carbon nano-onions (CNOs) were used as supports to immobilize alkaline phosphatase, horseradish peroxidase, and glucose oxidase. CNOs were first functionalized by oxidation to generate carboxylic groups on the surface followed by the covalent linking of using a soluble carbodiimide as coupling agent. The CNO–enzyme conjugates were characterized by transmission electron microscopy and Raman spectroscopy. Thermogravimetric analysis revealed a specific enzyme load of ～0.5?mg of protein per milligram of CNO. The immobilized enzymes showed enhanced storage stability without altering the optimum pH and temperatures. These properties make the prepared nanobiocatalyst of potential interest in biosensing and other biotechnological applications.     

Covalent immobilization of glucose oxidase onto poly(styrene-co-glycidyl methacrylate) monodisperse fluorescent microspheres synthesized by dispersion polymerization

In this study, micron-sized poly(styrene-co-glycidyl methacrylate) (PSt-GMA) fluorescent microspheres of 5.1microm in diameter were synthesized via dispersion polymerization of styrene and glycidyl methacrylate in the presence of 1,4-bis(5-phenyloxazol-2-yl) benzene (POPOP), which provided surface functional groups for covalent immobilization of enzymes. In an effort to study the biocompatibility of the microspheres' surface, glucose oxidase and beta-d-(+)-glucose were selected as a catalytic system for enzymatic assays. A colorimetric method was adopted in evaluating enzymatic activity by introducing horseradish peroxidase (HRP). Both the immobilization amount and the apparent activity of immobilized glucose oxidase from Aspergillus niger (GOD) were determined at different conditions. The results show that the immobilized enzymes retained approximately 28 to 34% activity, as compared with free enzymes, without pronounced alteration of the optimum pH and temperature. Kinetics studies show that the corresponding values of K(m) and V(max) are 23.2944 mM and 21.6450M/min.mg GOD for free enzymes and 35.1780 mM and 15.4799M/min.mg GOD for immobilized enzymes. The operational stability studies show that immobilized GOD could retain nearly 50% initial activity after being washed 20 times. The results suggest that the resultant PSt-GMA fluorescent microspheres provide a suitable surface for covalent immobilizing biomolecules; therefore, they have the potential of being used in fluorescence-based immunoassays in high-throughput screening or biosensors.     

Enhanced oxidation of bilirubin by an immobilized tri-enzyme system of glucose oxidase, bilirubin oxidase and horseradish peroxidase

Bilirubin oxidase was immobilized to nylon fibres. A tri-enzyme system composed of glucose oxidase, bilirubin oxidase and horseradish peroxidase was also immobilized to the fibres. Both immobilized systems were tested and it was found that the latter gave enhanced oxidation rates for bilirubin.     

Enhancement of oxygen absorption by magnetite-containing beads of immobilized glucose oxidase

Rates of oxygen absorption into glucose solutions were measured using an immobilized-enzyme reactor, in which magnetite-containing beads of immobilized glucose oxidase were moved by a revolving magnetic field to reduce the mass transfer resistances at the gas–liquid interface and around the bead. Data were also obtained for oxygen absorption into glucose solutions containing soluble or immobilized glucose oxidase (without magnetite), as well as for physical absorption of oxygen. The rates of physical absorption for the runs with the magnetite-containing beads increased because of mechanical stirring caused by spinning of the beads at the gas-liquid interface. In this case the experimental enhancement factors were found to be larger than those predicted on the basis of the film theory for gas absorption with a pseudo-first order reaction.     

Continuous conversion of sucrose to fructose and gluconic acid by immobilized yeast cell multienzyme complex

A multienzyme complex consisting of invertase, glucose oxidase, and catalase was reconstituted by binding glucose oxidase using concanavalin A (Con A) to the cell wall of Sacchararomyces cerevisiae, previously induced for maximal activities of invertase and catalase. The cell flocculate obtained was stabilized by entrapment in polyacrylamide using γ irradiation at 100 kR. This complex showed a shortening of the lag period and enhancement in gluconic acid production as compared to a similar mixture of soluble enzymes. The efficacy of the multienzyme complex has been compared with that of mixed multienzyme system composed of individually immobilized enzymes. The immobilized multienzyme complex in a continuous-flow stirred-tank reactor system could be operated for continuous conversion of sucrose to fructose and gluconic acid. The reactor system did not show any loss in efficiency in a continuous operation over 20 days.     

Covalent immobilization of enzymes within micro-aqueous organic media

Traditional covalent immobilization of enzymes was mostly operated within water phase. However, most of enzymes are flexible when they are in water environment, and the covalent reactions generally lead to complete or partial activity losing due to the protein conformational changes.This paper examined enzyme covalent immobilization operated in micro-aqueous organic media, to display the differences between two environments of immobilization within water and micro-aqueous organic solvent by activity and stability determination of the resulting immobilized enzymes. Catalase, trypsin, horseradish peroxidase, laccase and glucose oxidase have been employed as model enzymes. Results showed the thermal, pH and reusable stabilities of the micro-aqueous organic covalently immobilized enzymes were improved when compared with the immobilized enzymes within water. Micro-aqueous covalent immobilization showed a remarkable advantage in remaining the enzymes catalytic activity for all the five enzymes compared with the traditional water phase immobilization. And the optimum pH values for both immobilization within water and micro-aqueous organic media shifted slightly.     

The Application of Chemiluminescence of a Cypridina Luciferin Analog to Immobilized Enzyme Sensors

Chemiluminescence of a Cypridina luciferin analog, 2-methyl-6-phenyl-3,7-dihydro-imidazo[l,2-a]pyrazin-3-one, was applied to immobilized enzyme sensors. Xanthine oxidase, peroxidase, glucose oxidase, uricase and cholesterol oxidase were immobilized by using photo-crosslinkable resin prepolymer or ion-exchangeable cellulose beads. The immobilized enzyme sensor system was composed of a photoncounter and a test tube in which the immobilized enzyme membrane or particles were placed. A linear relation between the concentration of substrates and luminescence rate was obtained on a logarithmic scale. This immobilized enzyme sensor system could be used repeatedly. Hydrogen peroxide, xanthine and hypoxanthine were measured sensitively and rapidly within 100 sec. Glucose, cholesterol and uric acid were measured sensitively within 10 min but could be measured within 100 sec, although less sensitive. The detection limits for xanthine, hypoxanthine, hydrogen peroxide, glucose, cholesterol and uric acid were 0.02, 0.02, 0.2, 0.4, 2 and 2 μM, respectively. Concentrations of hypoxanthine in tuna muscle, and glucose and cholesterol in serum measured using this sensor system were comparable with those measured by the standard methods.     

Immobilization of glucose oxidase on silica-based supports

Glucose oxidase (beta-D-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4) was covalently coupled to silica-based supports containing aldehyde functional groups. The activity of the immobilized enzyme was about 1000 U/g support. The optimum pH of the catalytic activity was 5.5 for the soluble enzyme and 6.0 for the immobilized enzyme. With glucose as a substrate the Km value of the immobilized enzyme was higher than in case of the soluble enzyme. The immobilized enzyme was found to be more thermostable than the soluble one. The immobilization did not affect the stability of glucose oxidase against the denaturing effect of urea.     

The immobilization of enzymes on nylon structures and their use in automated analysis

1. Glucose oxidase (EC 1.1.3.4) and urease (EC 3.5.1.5) were covalently attached through glutaraldehyde to low-molecular-weight nylon powder. 2. Immobilized derivatives of glucose oxidase and urease were prepared by cross-linking the respective enzymes within the matrix of a nylon membrane. 3. An improved process is described for the immobilization of glucose oxidase and urease on the inside surface of partially hydrolysed nylon tube. 4. Automated analytical procedures are described for the determination of glucose with each of the three immobilized glucose oxidase derivatives and for the determination of urea with each of the three immobilized urease derivatives. 5. The efficiencies of the three immobilized enzyme structures as reagents for the automated determination of their substrates were compared.     

Multi-step reactions on microchip platform using nitrocellulose membrane reactor

A straightforward and effective method is presented for immobilizing enzymes on a microchip platform without chemically modifying a micro-channel or technically microfabricating a column reactor and fluid channel network. The proposed method consists of three steps: the reconstitution of a nitrocellulose (NC) membrane on a plane substrate without a channel network, enzyme immobilization on the NC membrane, and the assembly of another substrate with a fabricated channel network. As a result, enzymes can be stably and efficiently immobilized on a microchip. To evaluate the proposed method, two kinds of enzymatic reaction are applied: a sequential two-step reaction by one enzyme, alkaline phosphatase, and a coupled reaction by two enzymes, glucose oxidase and peroxidase, for a glucose assay.     

Reversible coupling of glucoenzymes on fluoride-sensitive FET biosensors based on lectin-glucoprotein binding

New reloadable biosensors were developed by affinity binding of glucoenzymes peroxidase and glucose oxidase on pF-sensitive field-effect transistors (Si/SiO₂/Si₃N₄/LaF₃ layers). The basic measuring principle of these ion sensitive field-effect transistors is the current change depending on the concentration of F⁻ ions. The immobilized glucoenzymes can be removed from the enzymatically inactive Concanavalin A basic membrane and fresh enzymes can be bound again. The principle of this reloadable biosensor is described in detail. Additionally, the biosensor was successfully integrated in a flow-injection analysis system for measuring glucose during a cultivation process.