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PubMed1976

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.

Glass↗
PubMed2000

Aryl-alcohol oxidase protein sequence: a comparison with glucose oxidase and other FAD oxidoreductases.

Aryl-alcohol oxidase (AAO), an FAD-dependent enzyme involved in lignin degradation, has been cloned from Pleurotus eryngii. The AAO protein is composed of 593 amino acids, 27 of which form a signal peptide. It shows 33% sequence identity with glucose oxidase from Aspergillus niger and lower homology with other oxidoreductases. The predicted secondary structures of both enzymes are very similar. For AAO, it is predicted to contain 13 putative alpha-helices and two major beta-sheets, each of the putative beta-sheets formed by six beta-strands. The ADP binding site and the signature-2 consensus sequence of the glucose-methanol-choline (GMC) oxidoreductases were also present. Moreover, residues potentially involved in catalysis and substrate binding were identified in the vicinity of the flavin ring. They include two histidines (H502 and H546) and several aromatic residues (Y78, Y92 and F501), as reported in other FAD oxidoreductases.

Alcohol Oxidoreductases↗
PubMed1987

Reconstitution of pyrroloquinoline quinone-dependent D-glucose oxidase respiratory chain of Escherichia coli with cytochrome o oxidase.

D-Glucose dehydrogenase is a pyrroloquinoline quinone-dependent primary dehydrogenase linked to the respiratory chain of a wide variety of bacteria. The enzyme exists in the membranes of Escherichia coli, mainly as an apoenzyme which can be activated by the addition of pyrroloquinoline quinone and magnesium. Thus, membrane vesicles of E. coli can oxidize D-glucose to gluconate and generate an electrochemical proton gradient in the presence of pyrroloquinoline quinone. The D-glucose oxidase-respiratory chain was reconstituted into proteoliposomes, which consisted of two proteins purified from E. coli membranes, D-glucose dehydrogenase and cytochrome o oxidase, and E. coli phospholipids containing ubiquinone 8. The electron transfer rate during D-glucose oxidation and the membrane potential generation in the reconstituted proteoliposomes were almost the same as those observed in the membrane vesicles when pyrroloquinoline quinone was added. The results demonstrate that the quinoprotein, D-glucose dehydrogenase, can reduce ubiquinone 8 directly within phospholipid bilayer and that the D-glucose oxidase system of E. coli has a relatively simple respiratory chain consisting of primary dehydrogenase, ubiquinone 8, and a terminal oxidase.

Electron Transport Complex IV↗
PubMed1975

Preparation of a modified glucose oxidase reagent for the polarographic determinationof glucose with the Beckman "glucose analyzer".

In this modification, glucose oxidase, 140-180 U/ml, is dissolved in acetate buffer (pH 6):ethanol (96%):glycerol (80:10:10 by vol). Iodine and molybdate are used as catalysts, formaldehyde and mercuric iodide as preservatives, and octanol as an antifoaming agent. The reagent is suitable for use in the Beckman "Glucose Analyzer", it is stable at room temperature for at least six months, it is sensitive, it has a lubricating effect, and its cost is less than a fifth as much as the "Certified" Beckman reagent. It is not suitable for the measurement of glucose in samples containing maltose, cellobiose, gentiobiose, starch, and glycogen.

Body Fluids↗
PubMed1975

The preparation of several new nylon tube-glucose oxidase derivatives and their incorporation into the "reagentless" automated analysis of glucose.

Nylon tube was activated by alkylation with dimethyl sulfate and used for the immobilization of glucose oxidase. Lysine, hexamethylene diamine and polyethylene imine were also attached to activated nylon tube, and these nylon tube-spacer derivatives were reactivated with either glutaraldehyde or ethyl adipimidate for the subsequent coupling of glucose oxidase. The activities of all of the different nylon tube-glucose oxidase derivatives were compared by their incorporation into standard Technicon automated analysis systems. Activities were measured either spectrophotometrically, by following the production of hydrogen peroxide using an acid/KI assay, or polarographically by following the decrease in the oxygen concentration using a flow-through oxygen electrode assembly. The activity and stability of all of the nylon tube-glucose oxidase derivatives was such that their use in the routine estimation of glucose levels was an attractive proposition.

Amines↗
PubMed2006

Preserved enzymatic activity of glucose oxidase immobilized on unmodified electrodes for glucose detection.

Glucose sensing electrodes have been realized by immobilizing glucose oxidase (GOx) on unmodified edge plane of highly oriented pyrolytic graphite (epHOPG) and the native oxide of heavily doped silicon (SiO2/Si). Both kinds of electrode show direct interfacial electron transfer due to the redox process of the immobilized GOx. The measured formal potential of the redox process agrees with that of the native enzyme, suggesting that the immobilized GOx has retained its enzymatic activity. The electron transfer rates of the GOx immobilized electrode are 2s(-1) for GOx/epHOPG electrode and 7.9s(-1) for GOx/SiO2/Si electrode, which are greater than those for which GOx is immobilized on modified electrodes, probably due to the fact that the enzyme makes direct contact to electrode surface. The preservation of the enzymatic activity of the immobilized GOx has been confirmed by observing the response of the GOx/epHOPG and GOx/SiO2/Si electrodes to glucose with a detection limit of 0.050 mM. The response signals the catalyzed oxidation of glucose and, therefore, confirms that the immobilized GOx retained its enzymatic activity. The properties of the electrode as a glucose sensor are presented.

Biosensing Techniques↗
PubMed1998

Glucose oxidase from Penicillium amagasakiense. Primary structure and comparison with other glucose-methanol-choline (GMC) oxidoreductases.

The complete amino acid sequence of glucose oxidase from Penicillium amagasakiense was determined by Edman degradation and mass spectrometry of peptide fragments derived from three different specific proteolytic digests and a cyanogen bromide cleavage. The complete sequence of each monomer comprises 587 amino acid residues, contains three cysteine residues, and seven potential N-glycosylation sites, of which at least five were confirmed to be glycosylated. Glucose oxidase from P. amagasakiense shows a high degree of identity (66%) and 79% similarity to glucose oxidase from Aspergillus niger, and is a member of the glucose-methanol-choline (GMC) oxidoreductase family. The tertiary structures of glucose oxidase from A. niger and cholesterol oxidase from Brevibacterium sterolicum were superimposed to provide a template for the sequence comparison of members of the GMC family. The general topology of the GMC oxidoreductases is conserved, with the exception of the presence of an active site lid in cholesterol oxidase and the insertion of additional structural elements in the substrate-binding domain of alcohol oxidase. The overall structure can be divided into five distinct sequence regions: FAD-binding domain, extended FAD-binding domain, flavin attachment loop and intermediate region, FAD covering lid, and substrate-binding domain. The FAD-binding and the extended FAD-binding domains are composed of several separate sequence regions. The other three regions each comprise a single contiguous sequence. Four major consensus patterns have been identified, including the nucleotide-binding consensus sequence close to their N-termini. The functions of the two motifs recently selected by the Genetics Computer Group, Madison, Wisconsin, as additional signature patterns of the GMC oxidoreductases are discussed. The other consensus patterns belong to either the FAD-binding or the extended FAD-binding domain. In addition, the roles of conserved residues are discussed wherever possible.

Amino Acid Sequence↗
PubMed1993

Induction of glucose oxidase, catalase, and lactonase in Aspergillus niger.

The induction of glucose oxidase, catalase, and lactonase activities was studied both in wild-type and in glucose oxidase regulatory and structural mutants of Aspergillus niger. The structural gene for glucose oxidase was isolated and used for Northern analysis and in transformation experiments using various gox mutations. Wild-type phenotype could be restored in the glucose oxidase-negative mutant (goxC) by transformation with the structural gene. We conclude, therefore, that the goxC marker which is located on chromosome 2 represents the structural gene of glucose oxidase. Glucose and a high oxygen level are necessary for the induction of all three enzyme activities in the wild-type strain and it was shown that both glucose and oxygen effects reflect regulation at the transcriptional level. The goxB mutation results in constitutive expression of all three activities although modulated to some extent by the carbon source. The goxE mutation only has an effect on lactonase and glucose oxidase expression and does not relieve the necessity for a high oxygen level. Catalase and lactonase could not be induced in the glucose oxidase-negative strain (goxC). Addition of H2O2 resulted in the induction of all three enzymes in the wild-type without glucose being present. The H2O2 induction is probably mediated by the goxB product. Besides the H2O2 induction there is still an effect of the carbon source on the induction. A model for induction of glucose oxidase, catalase, and lactonase in A. niger is discussed.(ABSTRACT TRUNCATED AT 250 WORDS)

Aspergillus↗
PubMed1975

[Comparison of blood sugar measured by Hoffman's, glucose oxidase and dextrostix technics during the oral glucose tolerance test].

The venous blood sugar has been measured in 384 adult subjects, between 20 and 60 years if agem during two oral glocose tolerance tests, with three methods: Hoffman's method, Glucose-oxidase method, and assay with reflectance meter (DRM). A good correlation was found with all methods (erg = 0,6 at 60 mn after load); however, the results given by the DRM were lower. So, in spite of its practical and economical advantages, the DRM can neverbe used for the diagnosis of an anomaly in the OGTT results. This method is a good one in emergency situations and in mass screening for diabetes.

Adult↗
PubMed1976

Immobilized glucose oxidase used in the continuous-flow determination of serum glucose.

We used a tubular glucose-oxidase wall reactor in the "AutoAnalyzer II" continuous-flow analytical system to determine glucose in blood serum. Sensitivity was high and wash characteristics were satisfactory with use of a 30-cm tube containing immobilized glucose oxidase. Results compared favorably with those of the conventional free-enzyme method. More than 25000 such assays can be performed with a single enzyme tube, which also shows long-term storage stability. Because of the steady-state chemistry 60 samples can be analyzed per hour. The linearity of the method is excellent and sample interaction from 5.0 to 1.0 g/liter is less than 5%. Results correlate well (greater than 0.993) with those obtained with both the neocuproine method used in the "SMA 12/60" multichannel analytical system and the free glucose oxidase method used in the AutoAnalyzer.

Autoanalysis↗
PubMed1976

Evaluation of a glucose oxidase procedure.

A commerical adaptation of Trinder's glucose oxidase procedure for glucose determination was evaluated by regression analysis. Excellent correlation was found between the glucose oxidase procedure and a hexokinase procedure. Good correlation was observed between an AutoAnalyzer I, the SMA 12/60, and a Beckman Glucose Analyzer, all utilizing variations of the glucose oxidase procedure. This combination provides a system that can handle screening, routine, and emergency requests in essentially the same manner and with comparable true glucose results. No interference was found with common anticoagulants and preservatives. Boehringer Mannheim Corporation's kit for glucose determinations appears to be of equivalent quality to the original method as outlined by Trinder and evaluated by others.

Anticoagulants↗
PubMed2006

[Recombinant Aspergillus niger glucose oxidase expressed in Trichoderma reesei].

It was expected that recombinant Aspergillus niger glucose oxidase could be expressed in Trichoderma reesei with stable activity. T. reesei CBHI promoter--CBHI ss. gene--A. niger glucose oxidase gene--T. reesei CBHI terminator--A. nidulans gpd promoter--E. coli Hygromycin B phosphotransferase gene--A. nidulans trpC terminator--pUC19 (pCBHGOD) vector was constructed in E. coli DH5alpha by PCR application and gene cloning methods. T. reesei QM9414 protoplast was transformed by T. reesei CBHI promoter-CBHI ss. Gene--A. niger glucose oxidase gene--T. reesei CBHI terminator-A. nidulans gpd promoter--E. coli Hygromycin B phosphotransferase gene--A. nidulans trpC terminator linear DNA fragment (CBHGOD fragment) that was made by digestion of pCBHGOD with Kpn I. T. reesei mutant clone with homologous recombinant A. niger glucose oxidase gene was selected by PCR method. Recombinant glucose oxidase was produced by mutant T. reesei strain under induction of wheat straw for 5 days. Recombinant glucose oxidase molecular mass was showed the same as native A. niger glucose oxidase standard from Sigma company by Western blot analysis. Recombinant glucose oxidase activity was 25u/mL in medium. The yield was 0.5 g/L in comparison with Sigma company glucose oxidase standard. There was no recombinant GOD degradation during Trichoderma reesei cultivation that was showed in Western blot analysis. Trichoderma reesei has capability to be a new recombinant host for Aspergillus niger GOD production.

Aspergillus niger↗
PubMed1994

Effects of sonicated eosinophils on the in vitro sensitivity of human lymphoma cells to glucose oxidase.

We report here that cultured human lymphoma cells in the absence of sonicated eosinophils are sensitive to killing by glucose oxidase (beta-D-glucose:oxygen-oxido reductase; EC 1.1.3.4) at concentrations as low as 0.025 microgram/ml, a level that can be rapidly attained in s.c. tumor implants in mice that receive a single nonlethal injection of enzyme. Multiple clonogenic assays were used to measure the survival of human lymphoma cell lines (H9 and ARH-77) cultured for 14 days in complete RPMI 1640 supplemented with exogenous glucose oxidase (0.025-2.5 micrograms/ml) or an immunoconjugate containing glucose oxidase (0.25-25 micrograms/ml) in the presence or absence of catalase (10 micrograms/ml) or an equal number of sonicated human eosinophils with or without supplemental 100 microM Br-, I-, or SCN-. In addition, we used an immunoassay to measure the concentration of glucose oxidase in s.c. implants of the Sp 2/0 myeloma tumor at 0-30 min after an i.v. injection of 50 micrograms of enzyme into 21 BALB/c mice. Doses of glucose oxidase as small as 0.025 microgram/ml killed more than 3 logs of tumor cells. Catalase completely inhibited, and sonicated human eosinophils partially inhibited, the killing by glucose oxidase or immunoconjugate, whereas supplemental halides had no effect. Glucose oxidase i.v. produced levels > 0.04 microgram/g of tumor for 30 min after injection with a peak concentration of 0.079 microgram/g of tumor within 5 min of injection. These results are important because certain human lymphomas contain extensive extracellular deposits of eosinophil peroxidase, thereby making these tumors potentially less susceptible to killing by otherwise therapeutic doses of glucose oxidase.

Drug Screening Assays, Antitumor↗
PubMedDate not supplied

[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.

Enzymes, Immobilized↗
PubMed2003

Alkaline treatment has contrasting effects on the structure of deglycosylated and glycosylated forms of glucose oxidase.

X-ray crystallographic studies on glucose oxidase showed a strong interaction between carbohydrate and protein moieties of the glycoprotein. However, experimental studies under physiological conditions reported no influence of carbohydrate moiety on the structural and functional properties of glucose oxidase. In order to demonstrate the role of carbohydrate moiety on the structure and stability, we carried out a detailed comparative study on the pH-induced structural changes in the native and deglycosylated forms of glucose oxidase. Our studies demonstrate that at physiological pH both forms of enzyme have very similar structural and stability properties. Acid denaturation also showed similar structural changes in both forms of the enzyme. However, on alkaline treatment contrasting effects on the structure and stability of the two forms of enzyme were observed. The glycosylated enzyme undergoes partial unfolding with decreased stability at alkaline pH; however, a compaction of native conformation and enhanced stability of enzyme was observed for the deglycosylated enzyme under similar conditions. This is the first experimental demonstration of the influence of carbohydrate moiety on structure and stability of glucose oxidase. The studies also indicate the importance of pH studies in evaluating the effect of carbohydrate moiety on the structural and stability properties of glycoprotein.

Carbohydrates↗
PubMed1999

Inactivation of glucose oxidase by diperoxovanadate-derived oxidants.

Inactivation of glucose oxidase occurred in the presence of bromide, vanadate, H(2)O(2), and phosphate (the bromide system), and this was prevented by NADH or phenol red, a bromine acceptor. Glucose oxidase present during the reaction between diperoxovanadate and a reduced form of vanadate, vanadyl (the vanadyl system), but not added after mixing the reactants, was inactivated, and this was accompanied by a loss of binding of the dye, Coomassie blue, to the protein. The transient intermediate of the type OVOOV(O(2)), known to form in these reactions and used in the oxidation of bromide ion and NADH, appears to be responsible for inactivating glucose oxidase. In both systems, the inactivation of the enzyme was prevented by histidine and DTT, known to quench singlet-oxygen. By direct measurement of 1270-nm emission of singlet-oxygen, its generation was demonstrated in the bromide system, and in the reaction of hypohalous acids with diperoxovanadate, but not in the vanadyl system. By themselves both hypohalous acids, HOCl and HOBr inactivated glucose oxidase, and their prior reaction with H(2)O(2) during which singlet-oxygen was released, protected the enzyme. The results provide support for possible oxidative inactivation of glucose oxidase by diperoxovanadate-derived oxidants.

Bromides↗
PubMed1979

Detergent-containing glucose oxidase reagent for use with the Beckman glucose analyzer.

We describe a modified glucose oxidase reagent for use in the polarographic determination of glucose with the Beckman "Glucose Analyzer." The reagent contains 1 mL/L of a surfactant (Brij-35 250 g/L solution) as the wetting agent instead of glycerol. Precipitation of components associated with the formulation recommended by Fischl et al. [Clin. Chem. 21, 760 (1975)] does not occur with this reagent. It can be used immediately after preparation. When compared to analytical performance of the commercially prepared reagent, the precision was unchanged by the modified reagent, but the upper limit of accurate response was diminished (7.5 g/L vs. 6.7 g/L). The modified reagent is less expensive than are commercially prepared reagents.

Autoanalysis↗
PubMed1991

15N- and 13C-NMR investigations of glucose oxidase from Aspergillus niger.

The apoprotein of glucose oxidase from Aspergillus niger was reconstituted with specifically 15N- and 13C-enriched FAD derivatives and investigated by 15N- and 13C-NMR spectroscopy. On the basis of the 15N-NMR results it is suggested that, in the oxidized state of glucose oxidase, hydrogen bonds are formed to the N(3) and N(5) positions of the isoalloxazine system. The hydrogen bond to N(3) is more pronounced than that to N(5) as compared with the respective hydrogen bonds formed between FMN and water. The resonance position of N(10) indicates a small decrease in sp2 hybridization compared to free flavin in water. Apparently the isoalloxazine ring is not planar at this position in glucose oxidase. Additional hydrogen bonds at the carbonyl groups of the oxidized enzyme-bound FAD were derived from the 13C-NMR results. A strong downfield shift observed for the C(4a) resonance may be ascribed in part to the decrease in sp2 hybridization at the N(10) position and to the polarization of the carbonyl groups at C(2) and C(4). The polarization of the isoalloxazine ring in glucose oxidase is more similar to FMN in water than to that of tetraacetyl-riboflavin in apolar solvents. In the reduced enzyme the N(1) position is anionic at pH 5.6. The pKa is shifted to lower pH values by at least 1 owing to the interaction of the FAD with the apoprotein. As in the oxidized state of the enzyme, a hydrogen bond is also formed at the N(3) position of the reduced flavin. The N(5) and N(10) resonances of the enzyme-bound reduced FAD indicate a decrease in the sp2 character of these atoms as compared with that of reduced FMN in aqueous solution. Some of the 15N- and 13C-resonance positions of the enzyme-bound reduced cofactor are markedly pH-dependent. The pH dependence of the N(5) and C(10a) resonances indicates a decrease in sp2 hybridization of the N(5) atom with increasing pH of the enzyme solution.

Aspergillus niger↗