Inactivation Of Glucose Oxidase Research Articles

Antibacterial Activity of Different Blossom Honeys: New Findings.

Antibacterial activity is the most investigated biological property of honey. The goal of this study was to evaluate the antibacterial activity of 57 Slovak blossom honeys against Staphylococcus aureus and Pseudomonas aeruginosa and investigate the role of several bioactive substances in antibacterial action of honeys. Inhibitory and bactericidal activities of honeys were studied to determine the minimum inhibitory and bactericidal concentrations. The contents of glucose oxidase (GOX) enzyme, hydrogen peroxide (H2O2), and total polyphenols (TP) were determined in honeys. We found that honey samples showed different antibacterial efficacy against the tested bacteria as follows: wildflower honeys > acacia honeys > rapeseed honeys. Overall antibacterial activity of the honeys was statistically-significantly correlated with the contents of H2O2 and TP in honeys. A strong correlation was found between the H2O2 and TP content. On the other hand, no correlation was found between the content of GOX and level of H2O2. Antibacterial activity of 12 selected honeys was markedly reduced by treatment with catalase, but it remained relatively stable after inactivation of GOX with proteinase-K digestion. Obtained results suggest that the antibacterial activity of blossom honeys is mainly mediated by H2O2 levels present in honeys which are affected mainly by polyphenolic substances and not directly by GOX content.

Investigation of structural stability and enzymatic activity of glucose oxidase and its subunits

Glucose oxidase (β-d-glucose:oxygen 1-oxidoreductase, EC 1.1.3.4) catalyzes the oxidation of β-d glucose utilizing molecular oxygen as an electron acceptor to produce d-glucono-1,5-lactoneand hydrogen peroxide, which has applications in food, biotechnology and medical industries. It was known that dimer form was considered to be active and monomer form has inactive conformation. However, there are no evidences at the molecular levels for Glucose oxidase (GOx) inactivation through dissociation. Here, using molecular dynamic simulation, it has been investigated for the first time that why dimer form of the enzyme is active. We have performed a series of molecular dynamics simulations at different forms of GOx (monomer and dimer with and without FAD cofactor). The analysis of tertiary structure showed that monomer is more unstable and has more deviation from the crystal structure. In contrast, dimer has a stable conformation during simulation. These results are in good agreement with experimental data about enzyme inactivation by dissociation. It was also found that when FAD is removed from monomer, it became more unstable in comparison with monomer containing cofactor. This shows essential role of FAD in both activity and stability of the enzyme. According to the MD simulation, enzyme inactivation is associated with changing in secondary structure at the interface. Interestingly, it was found that some secondary structures are destructed while some structures are formed in monomer upon dissociation. The analysis of active site structure during simulation revealed that both dissociation and release of the FAD influence on inactivation of GOx. This study provided novel insight to understand the mechanism of enzyme inactivation upon dissociation, which would be useful for rational enzyme design.

Protective effect of dextrans on glucose oxidase denaturation and inactivation

In the present study, the stabilizing effect of dextrans as additives on the denaturation and inactivation of glucose oxidase (GOD) was investigated. Three different molecular weighted dextrans (M(w) 17.5, 75, 188 kD) were used with different concentrations. Dramatically increased enzyme activities were measured after one hour of incubation of enzyme with additives between 25-40°C in water bath. Highest activity value was measured with 75 kDa molecular weighted dextran (in concentration 30% w/v) at pH 5. Dextran as an additive supplied a long shelf-life to the enzyme at 4°C. In the presence of the 75 kDa dextran, the enzyme was more stable and its activity was increased 2.7-fold at 30°C. In addition, dextran protected GOD against inactivation by a n-heptane/aqueous buffer-stirred system.

The mechanism of inactivation of glucose oxidase from Penicillium amagasakiense under ambient storage conditions

Glucose oxidase (GOx) from Penicillium amagasakiense has a higher specific activity than the more commonly studied Aspergillus niger enzyme, and may therefore be preferred in many medical and industrial applications. The enzyme rapidly inactivates on storage at pH 7.0–7.6 at temperatures between 30 and 40 °C. Results of fluorimetry and circular dichroism spectroscopy indicate that GOx inactivation under these conditions is associated with release of the cofactor FAD and molten globule formation, indicated by major loss of tertiary structure but almost complete retention of secondary structure. Inactivation of GOx at pH < 7 leads to precipitation, but at pH ≥ 7 it leads to non-specific formation of small soluble aggregates detectable by PAGE and size-exclusion chromatography (SEC). Inactivation of P. amagasakiense GOx differs from that of A. niger GOx in displaying complete rather than partial retention of secondary structure and in being promoted rather than prevented by NaCl. The contrasting salt effects may reflect differences in the nature of the interface between subunits in the native dimers and/or the quantity of secondary structure loss upon inactivation.

An ultrasonic inactivation of Aspergillus niger glucose oxidase in aqueous solutions

The inactivation of Aspergillus niger glucose oxidase (GO) was studied in 0.02 M phosphate-citrate buffer (PCB) at various pH, temperatures of 37-59 degrees C, and sonication with low frequency (27 kHz, LF-US) and high frequency (2.64 MHz, HF-US) ultrasound. The GO inactivation was characterized by the effective first-order inactivation rate constants k(in), k(in)*, and k(in)(us), reflecting the total, thermal, and ultrasonic inactivation components. The constants strongly depended on the pH and temperature of solution, GO concentration, and the presence of acceptors of the free radicals HO* -DMF, DMSO, ethanol, butanol, octanol, and mannitol, confirming that the active radicals formed in the ultrasonic cavitation field played an important role in the GO inactivation. The activation energy in the loss of GO catalytic activity considerably decreased when the enzyme solution was treated with LF-US or HF-US. The dissociative scheme of GO inactivation is discussed. Mannitol can be used for protection of GO from inactivation with LF-US or HF-US in the food industry and immunobiotechnology.

A New Bio-Inorganic Nanocomposite Membrane for Glucose-Modulated Release of Insulin

AbstractThis work focuses on the development of a new bio-inorganic nanocomposite glucose-responsive membrane to be applied as a single self-regulated platform for insulin delivery. Crosslinked bovine serum albumin (BSA)-based membranes were prepared containing impregnated pH-responsive poly(N-isopropyl acrylamide-co-methacrylic acid) nanoparticles (hydrogel NPs), glucose oxidase (GOx), catalase (CAT), with or without MnO2 NPs. The membrane acts as a glucose sensor and insulin release attenuator. In this system glucose is oxidized by GOx to produce gluconic acid, which regulates the permeability of the membrane to insulin. CAT and/or MnO2 NPs are introduced into the membrane in order to quench unwanted H2O2 produced by GOx turnover cycles, which can cause inactivation of GOx and toxicity. The glucose-modulated insulin release through the membrane is determined by alternating glucose concentration between 100 – 400 mg/dL (normal and hyperglycemic levels, respectively). The results show that the combination of CAT and MnO2 NPs in the membrane formulation leads to better efficiency in quenching the H2O2 and better long-term stability of GOx than using either alone. Very small amounts of insulin permeate though the membrane at the normal blood glucose level while a four-fold increase in the release rate is observed when glucose concentration is raised to a hyperglycemic level. The release rate of insulin drops when the glucose level is reduced to a normal value. These results demonstrate the self-regulated capability of the system.

Kinetics and Mechanism of Peroxyl Radical Reactions with Nitroxides

Cyclic nitroxides (>NO*) are stable radicals of diverse size, charge, lipophilicility, and cell permeability, which provide protection against oxidative stress via various mechanisms including SOD-mimic activity, oxidation of reduced transition metals and detoxification of oxygen- and nitrogen-centered radicals. However, there is no agreement regarding the reaction of nitroxides with peroxyl radicals, and many controversies in the literature exist. The question of whether nitroxides can protect by scavenging peroxyl radicals is important because peroxyl radicals are formed in biological systems. To further elucidate the mechanism(s) underlying the antioxidative effects of nitroxides, we studied by pulse radiolysis the reaction kinetics of piperidine, pyrrolidine, and oxazolidine nitroxides with several alkyl peroxyl radicals. It is demonstrated that nitroxides mainly reduce alkyl peroxyl radicals forming the respective oxoammonium cations (>N+=O). The most efficient scavenger of peroxyl radicals is 2,2,6,6-tetramethylpiperidine-N-oxyl (TPO), which has the lowest oxidation potential among the nitroxides tested in the present study. The rate constants of peroxyl reduction are in the order CH2(OH)OO*>CH3OO*>t-BuOO*, which correlate with the oxidation potential of these peroxyl radicals. The rate constants for TPO vary between 2.8x10(7) and 1.0x10(8) M-1 s-1 and for 3-carbamoylproxyl (3-CP) between 8.1x10(5) and 9.0x10(6) M-1 s-1. The efficacy of protection of nitroxides against inactivation of glucose oxidase caused by peroxyl radicals was studied. The results demonstrate a clear correlation between the kinetic features of the nitroxides and their ability to inhibit biological damage inflicted by peroxyl radicals.

Intracellular glucose oxidase from Penicillium funiculosum 46.1

A method for isolating extracellular glucose oxidase from the fungus Penicillium funiculosum 46.1, using ultrafiltration membranes, was developed. Two samples of the enzyme with a specific activity of 914-956 IU were obtained. The enzyme exhibited a high catalytic activity at pH above 6.0. The effective rate constant of glucose oxidase inactivation at pH 2.6 and 16 degrees C was 2.74 x 10(-6) s-1. This constant decreased significantly as pH of the medium increased (4.0-10.0). The temperature optimum for glucose oxidase-catalyzed beta-D-glucose oxidation was in the range 30-65 degrees C. At temperatures below 30 degrees C, the activation energy for beta-D-glucose oxidation was 6.42 kcal/mol; at higher temperatures, this parameter was equal to 0.61 kcal/mol. Kinetic parameters of glucose oxidase-catalyzed delta-D-glucose oxidation depended on the initial concentration of the enzyme in the solution. Glucose oxidase also catalyzed the oxidation of 2-deoxy-D-glucose, maltose, and galactose.

Peroxo-bridged divanadate as selective bromide oxidant in bromoperoxidation.

Diperoxovanadate is effective only in presence of free vanadate in vanadium-dependent bromoperoxidation at physiological pH. Peroxide in the form of bridged divanadate complex (VOOV-type), but not the bidentate form as in diperoxovanadate, is proposed to be the oxidant of bromide. In order to obtain direct evidence, peroxo-divanadate complexes with glycyl-glycine, glycyl-alanine and glycyl-asparagine as heteroligands were synthesized. By elemental analysis and spectral studies they were characterized to be triperoxo-divanadates, [V2O,(O2)3(peptide)3] x H2O, with the two vanadium atoms bridged by a peroxide and a heteroligand. The dipeptide seems to stabilize the peroxo-bridge by inter-ligand interaction, possibly hydrogen bonding. This is indicated by rapid degradation of these compounds on dissolving in water with partial loss of peroxide accompanied by release of bubbles of oxygen. The 51V-NMR spectra of such solutions showed diperoxovanadate and decavanadate (oligomerized from vanadate) as the products. Additional oxygen was released on treating these solutions with catalase as expected of residual diperoxovanadate. The solid compounds when added to the reaction mixtures showed transient, rapid bromoperoxidation reaction, but not oxidation of NADH or inactivation of glucose oxidase, the other two activities shown by a mixture of diperoxovanadate and vanadyl. This demonstration of peroxide-bridged divanadate as a powerful, selective oxidant of bromide, active at physiological pH, should make it a possible candidate of mimic in the action of vanadium in bromoperoxidase proteins.

Inactivation of Glucose Oxidase by Diperoxovanadate-Derived Oxidants

Inactivation of glucose oxidase occurred in the presence of bromide, vanadate, H2O2, 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(O2), 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 H2O2 during which singlet-oxygen was released, protected the enzyme. The results provide support for possible oxidative inactivation of glucose oxidase by diperoxovanadate-derived oxidants.

Mechanism of Inactivation of Glucoseoxidase by Hydrogenperoxide

Mechanism of Inactivation of Glucoseoxidase by Hydrogenperoxide