Characterization of the biochemical behavior of glucose oxidase entrapped in a polypyrrole film

This article reports the characterization of the biochemical behavior of glucose oxidase entrapped in polypyrrole. The immobilization of glucose oxidase in a polypyrrole film was performed by entrapment during the electropolymerization of pyrrole at a platinum electrode poised at 0.65 V vs. SCE in aqueous solution in a one-compartment electrochemical cell. Thin films of polypyrrole (0.11 mum) were obtained and the entrapped enzyme obeyed Michaelis kinetics, indicating no diffusional constraints of the substrate. Our results indicate that the entrapped glucose oxidase is more resistant to denaturation conditions such as alkaline pH and temperature (50 and 60 degrees C) than the soluble form of the enzyme. The autoinactivation constant for the entrapped enzyme was also determined in presence of 0.25M of glucose and was 6.19 x 10(-4) min(-1), i.e., corresponding to a half-life value of 20 h. The results reported here show clearly that polypyrrole matrix has a strong stabilizing effect on the stucture and on the activity of glucose oxidase.     

Preparation of immobilized enzymes by N-vinylpyrrolidone and the general properties of the glucoamylase gel

Immobilized glucoamylase, invertase, and β-galactosidase were prepared by using N-vinylpyrrolidone monomer (VP) under γ-ray irradiation. The enzyme-VP solutions were gelled by irradiation with 2.9 Mrad and the added enzymes were almost completely entrapped. Activity losses on entrapping were 55% for the VP-glucoamylase gel, and more than 90% in the case of VP-invertase and VP-β-galactosidase gels. No leakage of enzyme from these gels could be detected within 1 hr. The VP-glucoamylase gel was capable of hydrolyzing dextrin (mol wt 10,400) to glucose and the glucose equivalent was equal to that obtain able with native enzyme. The optimum temperature, heat stability, pH activity curve, and pH stability of VP-glucoamylase gel were slightly inferior to those of native enzyme, while Km was a little larger than that of native enzyme.     

Preparation and kinetic properties of gel entrapped urate oxidase.

Urate oxidase from hog liver (urate: oxygen oxidoreductase, EC 1.7.33) has been entrapped in a crosslinked 2-hydroxyethyl methacrylate gel with a 47% retention of activity. The kinetic behavior of the gel entrapped enzyme has been studied in a slurried tank reactor using uric acid as substrate. Internal diffusion effects were found to be negligible for particle sizes below 128 mum. A threefold increase in Km (app) was observed for the 128 mum particles and attributed to diffusional effects. The pH activity profile of the gel entrapped enzyme was bell-shaped at high substrate concentration and could be fitted to a titration curve of two ionizable groups, a basic group having a pK of 7.9 and an acidic group with a pK of 11.0. The gel entrapped enzyme showed excellent stability between pH 6.5 and 10.5.     

Optimization of a polypyrrole glucose oxidase biosensor

An amperometric glucose biosensor was fabricated by the electrochemical polymerization of pyrrole onto a platinum electrode in the presence of the enzyme glucose oxidase in a KCl solution at a potential of + 0·65 V versus SCE. The enzyme was entrapped into the polypyrrole film during the electropolymerization process. Glucose responses were measured by potentio-statting the enzyme electrode at a potential of + 0·7 V versus SCE in order to oxidize the hydrogen generated by the oxidation of glucose by the enzyme in the presence of oxygen. Experiments were performed to determined the optimal conditions of the polypyrrole glucose oxidase film preparation (pyrrole and glucose oxidase concentrations in the plating solution) and the response to glucose from such electrodes was evaluated as a function of film thickness, pH and temperature. It was found that a concentration of 0·3 M pyrrole in the presence of 65 U/ml of glucose oxidase in 0·01 M KCl were the optimal parameters for the fabrication of the biosensor. The optimal response was obtained for a film thickness of 0·17 μm (75 mC/cm²) at pH 6 and at a temperature of 313 K. The temperature dependence of the amperometric response indicated an activation energy of 41 kJ/mole. The linearity of the enzyme electrode response ranged from 1·0 mM to 7·5 mM glucose and kinetic parameters determined for the optimized biosensors were 33·4 mM for the K_m and 7·2 μA for the I_max. It was demonstrated that the internal diffusion of hydrogen peroxide through the polypyrrole layer to the platinum surface was the main limiting factor controlling the magnitude of the response of the biosensor to glucose. The response was directly related to the enzyme loading in the polypyrrole film. The shelf life and the operational stability of the optimized biosensor exceed 500 days and 175 assays, respectively. The substrate specificity of the entrapped glucose oxidase was not altered by the immobilization procedure.     

Immobilization of Mucor javanicus lipase by entrapping in alginate-silica hybrid gel beads with simultaneous cross-linking with glutaraldehyde

Mucor javanicus lipase was entrapped in alginate-silica hybrid gel beads with or without simultaneous cross-linking with glutaraldehyde. The activity and recovery of activity on immobilization of the enzyme entrapped in the hybrid beads were 1.4 and 1.7 times higher than those of the enzyme entrapped in the simple alginate beads. Entrapment with simultaneous cross-linking in the hybrid beads further improved the enzyme activity (1.6 times) and activity recovery (1.7 times) compared to those of the enzyme entrapped in the hybrid beads without simultaneous cross-linking. The leakage of the enzyme entrapped in the hybrid beads with simultaneous cross-linking was only 50% that of the enzyme entrapped in the simple alginate beads.     

Structural and biochemical properties of glycosylated and deglycosylated glucose oxidase from Penicillium amagasakiense

Glucose oxidase from Penicillium amagasakiense was purified to homogeneity by ion-exchange chromatography and deglycosylated with endoglycosidase H. On the basis of gas chromatography and sodium dodecyl sulphate/polyacrylamide gel electrophoretic (SDS-PAGE) analyses, the protein-bound high-mannose-type carbohydrate moiety corresponded to 13% of the molecular mass of glycosylated glucose oxidase. A total of six N-glycosylation sites per dimer were determined from the N-acetylglucosamine content. The enzymatically deglycosylated enzyme contained less than 5% of the original carbohydrate moiety. A molecular mass of 130 kDa (gel filtration) and 133 kDa (native PAGE) was determined for the dimer and 67 kDa (SDS-PAGE) for the monomer of the deglycosylated enzyme. The N-terminal sequence, which has not been published for glucose oxidase from P. amagasakiense to date and which showed less than 50% homology to the N terminus of glucose oxidase from Aspergillus niger, and the amino acid composition were not altered by the deglycosylation. Deglycosylation also did not affect the kinetics of glucose oxidation or the pH and temperature optima. It also did not increase the susceptibility of the enzyme to proteolytic degradation. However, deglycosylated glucose oxidase exhibited decreased pH and thermal stability. The thermal stability of both enzymes was shown to be dependent on the buffer concentration and was enhanced by certain additives, particularly 1 M (NH₄)₂SO₄, which stabilised glucose oxidase 100- to 300-fold at 50 °C and pH 7–8, and 2 M KF, which stabilised the enzyme up to 36-fold at 60 °C and pH 6. In sodium acetate buffer, changes in pH (4–6) affected the affinity for glucose but had no effect on the V _max of the reaction. In contrast, in TRIS buffer, pH 8, a 10-fold decrease in V _max and a 2-fold decrease in K _m were observed. Received: 8 October 1996 / Received revision: 14 January 1997 / Accepted: 17 January 1997     

Determination Method for l-Ascorbic Acid in Foods with Immobilized Ascorbate Oxidase

Pumpkin (Cucurbita moschata) ascorbate oxidase was entrapped within 6% (w/v) Ca-alginate gel beads, and then the beads were treated with 1% (w/v) glutaraldehyde for 20 hr at 4°C. The immobilized ascorbate oxidase was much more stable than the free form. Under the optimum conditions, the immobilized enzyme remained fully active for 3 months and after 50 assays. A linear relationship was found between immobilized ascorbate oxidase activity and l-ascorbic acid concentration in the range of 2 ~ 20 μg/ml. The immobilized preparation could be employed for the simple and rapid determination of l-ascorbic acid in foods.     

Investigation of concentration profiles inside operating biocatalytic sensors with scanning electrochemical microscopy (SECM)

Scanning electrochemical microscopy (SECM) with amperometric or potentiometric measuring tips was used to investigate biocatalytic reactions inside the enzyme layer of a biosensor during its operation. The well known glucose oxidase catalyzed oxidation of glucose has been selected for the studies. Local, instantaneous concentration of dissolved oxygen and hydrogen peroxide was studied observing the amperometric current while miniaturized potentiometric tip served for local pH measurements. Liquid enzyme layer immobilized with Cellophane membrane or cross linked polyacrilamide gel membrane containing entrapped enzyme served for biocatalytic media in the SECM imaging. Local maximum of H(2)O(2) and minimum of O(2) profiles were found at approximately 200 microm far from the substrate/enzyme layer boundary. From the experimental findings guidelines to design well functioning biocatalytic sensors could be concluded. The concentration profiles obtained with SECM techniques were compared with the results of simple model calculations carried out with the method of finite changes. Most of earlier made SECM studies dealing with enzyme reactions imaged the electrolyte being in contact with the immobilized enzyme. The data in our investigation, however, were collected inside the working catalytic layer.     

Purification and Properties of Pyranose Oxidase from Coriolus versicolor

Coriolus versicolor KY2912 grown on a medium containing glucose, sucrose or glycerol produced pyranose oxidase. Pyranose oxidase (glucose-2-oxidase) was purified by HPA-75 chromatography, Sepharose 4B and Sephadex G-100 gel filtration, and hydroxyapatite chromatography. The purified enzyme preparation showed a single protein band on acrylamide gel electrophoresis. The highest activity was obtained when D-glucose was employed as substrate and molecular oxygen as electron acceptor. The enzyme was most active at pH 6.2 and 50°C, stable in the pH region between 5.0 and 7.4, and the activity was completely lost above 70°C. The activity was inhibited by Ag⁺ , Cu²⁺ and PCMB. The enzyme contained FAD covalently bound to the polypeptide chain. The enzyme consisted of identical subunits with a molecular weight of 68,000, and showed a total molecular weight of 220,000.     

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     

Fabrication of sucrose biosensor based on single mode planar optical waveguide using co-immobilized plant invertase and GOD

In present studies, the new optical sensing platform based on optical planar waveguide (OPWG) for sucrose estimation was reported. An evanescent-wave biosensor was designed by using novel agarose–guar gum (AG) biopolymer composite sol–gel with entrapped enzymes (acid invertase (INV) and glucose oxidase (GOD)). Partially purified watermelon invertase isolated from Citrullus vulgaris fruit (specific activity 832 units mg⁻¹) in combination with GOD was physically entrapped in AG sol–gel and cladded on the surface of optical planar waveguide. Na⁺–K⁺ ion-exchanged glass optical waveguides were prepared and employed for the fabrication of sucrose biosensor. By addressing the enzyme modified waveguide structure with, the optogeometric properties of adsorbed enzyme layer (12 μm) at the sensor solid–liquid interface were studied. The OPWG sensor with short response time (110 s) was characterized using the 0.2 M acetate buffer, pH 5.5. The fabricated sucrose sensor showed concentration dependent linear response in the range 1 × 10⁻¹⁰ to 1 × 10⁻⁶ M of sucrose. Lower limit of detection of this novel AG–INV–GOD cladded OPWG sensor was found to be 2.5 × 10⁻¹¹ M sucrose, which indicates that the developed biosensor has higher sensitivity towards sucrose as compared to earlier reported sensors using various transducer systems. Biochips when stored at room temperature, showed high stability for 81 days with 80% retention of original sensitivity. These sucrose sensing biochips showed good operational efficiency for 10 cycles. The proper confinement of acid invertase and glucose oxidase in hydrogel composite was confirmed by scanning electron microscopy (SEM) images. The constructed OPWG sensor is versatile, easy to fabricate and can be used for sucrose measurements with very high sensitivity.     

Inactivation of Cytochrome-c with Glucose Oxidase

A novel reaction of cytochrome-c from the horse heart with the enzyme glucose oxidase from Aspergillus niger (EC 1.1.3.4), in acidic media is described. Glucose oxidase is able to induce a rapid, profound and irreversible physico-chemical change in cytochrome-c, under anaerobic conditions and in the presence of glucose. The initial rate of reaction is almost independent of the concentration of enzyme and glucose. The striking feature of this reaction is the fact that the reaction proceeds efficiently even below a concentration of 10?nM enzyme.     

Biosensors based on acrylic microgels: a comparative study of immobilized glucose oxidase and tyrosinase

Acrylic microgels are proposed as enzyme immobilizing support in amperometric biosensors. Two enzymes, glucose oxidase and tyrosinase, were entrapped in this matrix and their behaviour is compared. The optimum cross-linking of the polymeric matrix required to retain the enzyme, and to allow the diffusion of the substrate is different for each enzyme, 3.2% for glucose oxidase and 4.5% for tyrosinase. The effect of pH and temperature on the biosensor responses has been studied by experimental design methodology and predictions have been compared with independently performed experimental measurements. A quadratic effect of the variables studied (pH and T) on the biosensor response and the small or null interaction between them was confirmed. The pH results obtained with both methods are coincident revealing an reversible effect on the enzyme. However, the temperature optimum value obtained by experimental design was 10 degrees C lower as a result of an activity decay due to irreversible thermal denaturation of both enzymes.     

PURIFICATION AND CHARACTERIZATION OF GLYCOLATE OXIDASE FROM THE BROWN ALGA SPATOGLOSSUM PACIFICISM (PHAEOPHYTA)1

Glycolate oxidase was purified to apparent homogeneity from the brown alga Spatoglossum pacificum Yendo. The 1326-fold purified glycolate oxidase enzyme exhibited a specific activity of 22. 4 micromoles glyoxylate formed ·min^?1·mg protein^?1. The molecular weight of the native enzyme was estimated to be 230,000 by gel filtration. The subunit molecular weight of the enzyme was determined to be 49,000 by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, suggesting that the native enzyme is a tetramer. There were two absorption peaks at 345 and 445 nm, indicating that glycolate oxidase is a flavoprotein. This enzyme had a high isoelectric point (pI 9.6) and a high pH optimum (pH 8.3). The K_m values for glycolate and l -lactate were 0.49 and 5.5 mM, respectively. This enzyme also had a broad specificity for other straight-chain α-hydroxy acids but not for β-hydroxyacids. Cyanide, azide, N-ethylmaleimide, and p-chloromercuribenzoic acid did not affect the enzyme, whereas 2-pyridylhydroxymethanesulfonic acid strongly inhibited it. These properties of glycolate oxidase from the brown alga S. pacificum are similar to the properties of the glycolate oxidasesfrom higher plants. Polyclonal antibodies raised against the polypeptide fragment of Spatoglossum glycolate oxidase could recognize glycolate oxidase from Spinacia oleracea L., although the cross-reactivity was weak. The N-terminal sequence of two internal polypeptide fragments of the enzyme from S. pacificum showed a high degree of similarity to that of glycolate oxidase from higher plants. These results suggest that glycolate oxidase from higher plants and brown algae share the same ancestral protein.     

Improvement of the stability of glucose oxidase via encapsulation in sodium alginate–cellulose sulfate–poly(methylene-co-guanidine) capsules

Encapsulation of glucose oxidase (GOD) in polyelectrolyte complex capsules and its influence on properties of the enzyme is reported. The immobilization of GOD in the capsules made of sodium alginate (SA), cellulose sulfate (CS), poly(methylene-co-guanidine) (PMCG), CaCl₂ and NaCl (GOD–SA–CS/PMCG capsules) was achieved using a one-step highly reproducible encapsulation protocol which was monitored by a Electrospray Ionization-Mass Spectrometry (ESI-MS). A leakage of the enzyme from the capsules was negligible. Encapsulated GOD exhibited higher thermostability, wider range of pH optimum and improved storage stability in comparison with free GOD. The 92% retained activity by the encapsulated GOD after 45 biooxidation cycles was markedly higher than that of the GOD entrapped in calcium pectate gel beads showing no activity after 12 cycles. Optimization of conditions of oxygen supplementation resulted in increased oxygen availability within the GOD–SA–CS/PMCG capsules. Oxygen supplementation was accompanied with a mild decrease in the mechanical resistance of the SA–CS/PMCG capsules.     

Stabilization ofd-amino-acid oxidase fromTrigonopsis variabilis by manganese dioxide

Stabilization of immobilized D-amino-acid oxidase was achieved as follows. Yeast Trigonopsis variabilis producing D-amino-acid oxidase was used to deaminate cephalosporin C to glutaryl-7-aminocephalosporanic acid. Permeabilized cells were co-immobilized with manganese dioxide by entrapment in (poly)acrylamide gel so that hydrogen peroxide, liberated in the reaction, could be partially deactivated and both the enzyme and the substrate could be stabilized. Activity of entrapped cells was determined by HPLC and enzyme flow microcalorimetry. The process was evaluated in terms of activity, immobilization yield, storage stability and oxo-product formation by immobilized preparations. The storage stability of immobilized biocatalysts with MnO2 was nearly doubled and production of 2-oxoadipyl-7-aminocephalosporanic acid was 2-3-fold higher than by entrapped cells without MnO2. Glutaryl-7-aminocephalosporanic acid can be easily obtained from the resulting oxo-product by a non-enzymic reaction via externally added hydrogen peroxide.     

Improvement of the stability of glucose oxidase via encapsulation in sodium alginate–cellulose sulfate–poly(methylene-co-guanidine) capsules

Encapsulation of glucose oxidase (GOD) in polyelectrolyte complex capsules and its influence on properties of the enzyme is reported. The immobilization of GOD in the capsules made of sodium alginate (SA), cellulose sulfate (CS), poly(methylene-co-guanidine) (PMCG), CaCl₂ and NaCl (GOD–SA–CS/PMCG capsules) was achieved using a one-step highly reproducible encapsulation protocol which was monitored by a Electrospray Ionization-Mass Spectrometry (ESI-MS). A leakage of the enzyme from the capsules was negligible. Encapsulated GOD exhibited higher thermostability, wider range of pH optimum and improved storage stability in comparison with free GOD. The 92% retained activity by the encapsulated GOD after 45 biooxidation cycles was markedly higher than that of the GOD entrapped in calcium pectate gel beads showing no activity after 12 cycles. Optimization of conditions of oxygen supplementation resulted in increased oxygen availability within the GOD–SA–CS/PMCG capsules. Oxygen supplementation was accompanied with a mild decrease in the mechanical resistance of the SA–CS/PMCG capsules.     

Behavior of glucose oxidase immobilized in various electropolymerized thin films

Glucose oxidase was immobilized by electropolymerization into films of polyaniline, polyindole, polypyrrole, poly(o-phenylediamine), and polyaniline crosslinked with p-phenylenediamine. The kinetics and the behavior of the entrapped enzyme toward elevated temperature, organic solvent denaturation, and pH were investigated, along with the response of the films to electroactive species such as acetaminophen, ascorbate, cysteine, and uric acid. For most of the films, linearity to glucose extended from 7 to 10 mM. The poly(o-phenylenediamine)/glucose oxidase film gave the best signal/noise ratio and polypyrrole/glucose oxidase film gave the most reproducible current responses. No significant shift of the optimum reaction pH (5.5), except for polypyrrole (5.0), was observed after immobilization of glucose oxidase in the various films. Enzymatic activity decreased rapidly when pH was raised above 7.5. Thermodeactivation studies at 55 degrees , 60 degrees , and 65 degrees C have shown polypyrrole/and poly(o-phenylediamine)/glucose oxidase films to be the most resistant enzymatic films. Poly(o-phenylenediamine) films offered the best protection against glucose oxidase deactivation in hexane, chloroform, ether, THF, and acetonitrile when compared with the other electropolymerized films. All the enzymatic films exhibited permselection toward electroactive species. (c) 1996 John Wiley & Sons, Inc.     

Entrapment of a Trigonopsis variabilis D‐amino acid oxidase variant F54Y for oxidative deamination of cephalosporin C

Trigonopsis variabilis D ‐amino acid oxidase (TvDAAO) is an enzyme used in the industrial bioconversion of cephalosporin C (CPC) into 7‐aminocephalosporanic acid, a crucial biosynthetic nucleus for a wide spectrum of semi‐synthetic cephem antibiotics. Using homology modeling and site‐directed mutagenesis, we have previously shown that the TvDAAO variant F54Y possesses improved catalytic activity and thermostability. To further explore its industrial application, the conditions for immobilization of the enzyme were examined in the present investigation. The results showed that entrapment in a calcium alginate (Ca‐alginate) matrix using 2% alginate, 500 mM CaCl₂, and 15 min stabilization appeared to be optimal for the immobilization of F54Y. The entrapped enzyme allowed complete CPC conversion. The entrapped enzyme also showed good operational stability and retained at least 90% of its original activity after 20 reaction cycles. To conclude, the entrapment of F54Y in Ca‐alginate appeared to be a simple and efficient biocatalysis system with potential application in the antibiotics industry.     

Glucose sensing based on the intrinsic fluorescence of sol-gel immobilized yeast hexokinase

In this study, we investigated measurements of the intrinsic fluorescence of yeast hexokinase as an assay for glucose and immobilization of the enzyme in a silica sol-gel matrix as a potential in vivo glucose sensor for use in patients with diabetes. The intrinsic fluorescence of hexokinase in solution (excitation=295 nm, emission=330 nm) decreased by 23% at a saturating glucose concentration of 1 mM (Kd=0.3 mM), but serum abolished the glucose-related fluorescence response. When entrapped in tetramethylorthosilicate-derived sol gel, hexokinase retained activity, with a 25% maximal glucose-related decrease in intrinsic fluorescence, and the saturation point was increased to 50 mM glucose (Kd=12.5 mM). The glucose response range was increased further (to 120 mM, Kd=57 mM) by a covering membrane of poly(2-hydroxyethyl) methacrylate. Unlike free enzyme, the fluorescence responses to glucose with sol-gel immobilized hexokinase, with or without covering membrane, were similar for buffer and serum. We conclude that fluorescence monitoring of sol-gel entrapped yeast hexokinase is a suitable system for development as an in vivo glucose biosensor.