Short-Lived Radical Intermediates in the Photochemistry of Glucose Oxidase.

Glucose oxidase is a flavoprotein that is relatively well-studied as a physico-chemical model system. The flavin cofactor is surrounded by several aromatic acid residues that can act as direct and indirect electron donors to photoexcited flavin. Yet, the identity of the photochemical product states is not well established. We present a detailed full spectral reinvestigation of this issue using femtosecond fluorescence and absorption spectroscopy. Based on a recent characterization of the unstable tyrosine cation radical TyrOH•+ , we now propose that the primary photoproduct involves this species, which was previously not considered. Formation of this product is followed by competing charge recombination and radical pair stabilization reactions that involve proton transfer and radical transfer to tryptophan. A minimal kinetic model is proposed, including a fraction of TyrOH.+ that is stabilized up to the tens of picoseconds timescale, suggesting a potential role of this species as intermediate in biochemical electron transfer reactions.

Stimulation of glucose oxidase with white linearly polarized light.

Glucose oxidase (GOD) was illuminated with white linearly polarized light (WLPL). The enzyme was illuminated at room temperature in separate vessels then admixed to a reactor filled with D-glucose. The illumination of the enzyme for 60 min at 25-30 degrees C and pH 6.5-7.0 provided its superior stimulation as proven in the oxidation of beta-D-glucose. Lyophilization of the illuminated enzyme reduced its activity by, approximately, 30%.

Comparison of kinetic profile of free and immobilized glucose oxidase, immobilized on low-density polyethylene using UV polymerization.

Bioactive packaging is an important area of active packaging in which an active component is incorporated into the food contact surface of the package to interact with the food components without itself migrating into the food. Embedding bioactivity in a UV polymerizable resin is a novel and versatile technique for incorporating bioactive components into food packaging. In a previous article, glucose oxidase was immobilized in a packaging material using a UV curable resin. The relevance of this model system for deoxygenation of fruit juices was discussed. Though the technique efficiently immobilized enzymes in packaging material, during polymerization and immobilization the catalytic ability of the enzyme was not specifically explored. This article compares and contrasts the catalytic ability in terms of the kinetic profile of free and immobilized enzyme for the same model system: deoxygenation of juices. Kinetic behavior of immobilized and free glucose oxidase enzyme was evaluated at both shelf stable (room temperature) and refrigerated storage conditions to simulate the actual package life. It was observed that both the free enzyme and the immobilized enzymes follow the Michaelis-Menten kinetics model. There was no significant difference between the catalytic ability (k(cat)/K(m)) of free and immobilized enzymes at treatment temperatures (30, 25, and 10 degrees C); however, at refrigeration temperature (5 degrees C), the values for free enzyme were significantly higher than the immobilized enzyme.

Photoinhibition of manganese enzymes: insights into the mechanism of photosystem II photoinhibition.

Evidence has recently been presented that photoinhibition of photosystem II (PSII) is triggered by absorption of light by the oxygen-evolving manganese cluster. To get insight into the effects of light on enzymes containing manganese or other transition metal cofactors, the photosensitivities of Mn catalase, Mn superoxide dismutase, the haem (Fe)-containing bovine liver catalase, and CuZn superoxide dismutase were investigated. Glucose oxidase was studied as an example of an enzyme that does not have a metal cofactor. Sensitivities of these five enzymes to UVC, UVA, and visible light were compared in anaerobic conditions. The Mn(III)-oxo-Mn(III)-containing Mn catalase was found to be more sensitive to both visible and UV light than bovine liver catalase. Furthermore, the action spectrum of photoinhibition of Mn catalase was found to be fairly similar to that of photoinhibition of PSII. The Mn(II)-containing Mn superoxide dismutase was sensitive to UVC light and somewhat sensitive to UVA light, while only UVC light caused some inhibition of CuZn superoxide dismutase. Glucose oxidase was the least photosensitive of the enzymes studied. The photosensitivity of Mn enzymes supports the hypothesis that the oxygen-evolving manganese complex of PSII can be damaged by UV and visible light absorbed by its Mn(III) or Mn(IV) ions.

Immobilization and enzymatic activity of glucose oxidase on polystyrene surface modified with ozone aeration and UV irradiation in distilled water and/or aqueous ammonia solution.

Adsorption condition and enzymatic activity of glucose oxidase (GOD) on polystyrene (PS) film surfaces modified with ozone aeration and UV irradiation (O3/UV) treatment were investigated. The total amount of GOD immobilized on the PS film modified with the O3/UV treatment in distilled water (PS-W film) was approximately twice as large as that on the film treated in an aqueous ammonia solution (PS-A film), whereas the specific activity of GOD on the PS-A film was four times higher than that on the PS-W film. In contrast, no enzymatic activity of GOD on the non-treated PS film was observed because of irreversible denaturation of the adsorbed GOD. We therefore conclude that the PS films modified by the O3/UV treatment in the aqueous media are effective in immobilizing GOD.

Photochemical immobilization of proteins on microwave-synthesized photoreactive polymers.

We report a rapid and versatile procedure for the preparation of photoreactive polymers and light-induced immobilization of proteins onto such polymers. Photoreactive controlled-pore glass, silica gel, glass slide, and polystyrene microtiter plate are prepared in 40-60s by microwave irradiation of the respective amino polymers and 1-fluoro-2-nitro-4-azidobenzene. Azido group, now part of the polymer, yields highly reactive nitrene under ultraviolet (UV) light at 365 nm. Thus, when photoreactive polymer and horseradish peroxidase or glucose oxidase are exposed to UV light, the reactive nitrene immobilizes the protein molecules in 10 to 20 min through covalent bonding. As nitrene has a property of inserting into C-H bond, the method may find potential applications for immobilization of biomolecules irrespective of their functional groups.

Evaluation of the inhibiting effects from exposure to microwaves on the respiratory activity of yeast cells or on enzyme activity.

Microwaves are used in medical applications, so their eventual toxicity effects must be carefully evaluated. An integral toxicity test, based on the monitoring of the respiratory activity of yeast cells, is proposed to evaluate the damage from microwave exposure. Different exposure times and microwave powers were considered. On supposing that the damages occur at enzymatic levels, the inhibiting effects of microwave exposure on two enzymes (glucose oxidase (GOD) and superoxide dismutase (SOD)), assumed like models as present in the human organism, was evaluated.

Surface functionalization of polypyrrole film with glucose oxidase and viologen.

A surface modification technique was developed for the functionalization of polypyrrole (PPY) film with glucose oxidase (GOD) and viologen moieties. The PPY film was first graft copolymerized with acrylic acid (AAc) and GOD was then covalently immobilized through the amide linkage formation between the amino groups of the GOD and the carboxyl groups of the grafted AAc polymer chains in the presence of a water-soluble carbodiimide. Viologen moieties could also be attached to the PPY film via graft-copolymerization of vinyl benzyl chloride with the PPY film surface followed by reaction with 4,4'-bipyridine and alpha,alpha'-dichloro-p-xylene. X-ray photoelectron spectroscopy (XPS) was used to characterize the PPY films after each surface modification step. Increasing the AAc graft concentration would allow a greater amount of GOD to be immobilized but this would decrease the electrical conductivity of the PPY film. The activity of the immobilized GOD was compared with that of free GOD and the kinetic effects were also studied. The immobilized GOD was found to be less sensitive to temperature deactivation as compared to the free GOD. The results showed that the covalent immobilization technique offers advantages over the technique involving the entrapment of GOD in PPY films during electropolymerization. The presence of viologen in the vicinity of the immobilized GOD also enabled the GOD-catalyzed oxidation of glucose to proceed under UV irradiation in the absence of O(2).

[Optimization of the conditions of immobilization of enzymes in a photopolymeric membrane]. / Optimizatsiia uslovii immobilizatsii fermentov v fotopolimernoi membrane.

The residual activity of enzymes immobilized in the membrane on the basis on 1-vinyl-2-pyrrolidinone as photopolymerizable composition is studied. It is established, that under conditions of the immobilization at 20 degrees C the residual activity glucoseoxidase is about 35% from a initial level, horseredish peroxidase and urease from Jeack beans--42% and 20%, respectively. In case of an immobilization of beta-glucoseoxidase -50 degrees C it reaches almost 50% from a initial level. It was investigated the influence of different sources of UV-radiation and different substances on stability of the enzymes in the composition and in the immobilization matrix at storage. Dynamic of changes of enzyme activity at the photoimmobilization was characterized, and also the requirements for providing of its maximal storage was selected.

Excited-state dynamics of fully reduced flavins and flavoenzymes studied at subpicosecond time resolution.

The photophysics of the fully reduced states of a number of flavins (flavin mononucleotide, flavin adenine dinucleotide and 3-N-methyllumiflavin) and flavoenzymes (glucose oxidase from Aspergillus niger and the flavodehydrogenase component isolated from flavocytochrome b2) was studied using subpicosecond laser excitation at lambda = 312 nm. The prompt transient absorption spectra (measured from 400 to 850 nm) were all closely similar in the case of the free flavins in aqueous solution. The decay of the transient absorbance obeyed biexponential kinetics with a fast component of lifetime ranging from 4 to 130 ps and a slower phase with a lifetime above 1 ns. The spectral structure changed appreciably during the rapid decay phase. In contrast, in the case of the enzymes only a very slight decay was apparent over the probed time interval (1 ns) and the shape of the spectrum remained unchanged. It is proposed that the two transient spectra appearing in the free flavins correspond to two conformations differing by their degree of nonplanarity, whereas in the flavoenzymes only one conformation is possible.

Matrix entrapment of glucose oxidase by gamma irradiation.

In this study, matrix entrapment of the enzyme glucose oxidase was achieved through gamma irradiation of monomers N-vinyl pyrrolidone, 2-hydroxyethyl methacrylate and their mixture. To test the effect of radiation on entrapment efficiency, retention of activities and properties of the system, duration and temperature were varied. The reusability of the resultant products was tested. It was generally found that inclusion of the hydrophilic monomer N-vinyl pyrrolidone into the matrix increased the water content, entrapment efficiency and enzyme activity. gamma irradiation did not have a detrimental effect on the activity of pure enzyme when exposure was at -196 degrees C, but this radioresistance was less at higher temperatures. Infrared spectroscopy indicated that there was no chemical binding between the matrix and the enzyme. Repeated use studies revealed a gradual loss of activity in all of the samples tested for 10 runs.

Effect of irradiation on immobilized enzymes compared with that on enzymes in solution.

Glucose oxidase and catalase were immobilized by attaching them to nylon fibers that had been treated with triethyloxonium-tetrafluoroborate, diaminohexane and glutardialdehyde according to Morris, Campbell and Hornby (1975). This method assures that the enzymes are bound to a side chain of the polyamide structure. Enzyme activity (as measured by the O2-uptake and by microcalorimetry) was found to be unchanged after 2 years. The apparent Km-constants of the immobilized enzymes with glucose were the same as those for enzymes in solution. GOD and catalase immobilized in poly(acrylamide) gel had the same Km-value. Despite the high stability during storage, the radiation induced inactivation of enzymes immobilized on gel or chromosorb, an inorganic carrier, was of the same order of magnitude as that of the dissolved enzymes. The enzymes bound to nylon fibers showed a higher radiation sensitivity. This might have been caused by an additional attack on the binding site of the carrier.