Analysis and modeling of the ferulic acid oxidation by a glucose oxidase-peroxidase association. Comparison with a hexose oxidase-peroxidase association

A commercial glucose oxidase (GOX) from Aspergillus niger was partially characterized. The enzyme exhibited a two-step transfer mechanism, and the kinetic constants toward glucose and oxygen were determined. Under conditions similar to dough making (glucose concentration and pH), GOX does not exhibit maximum activity. A hexose oxidase (HOX) from Chondrus crispus was partially characterized as well. The HOX activity is not far from the optimum in the kneading conditions (pH and glucose concentration). A peroxidase (POD) purified from wheat germ was used to oxidize ferulic acid in the presence of GOX or HOX. Hydrogen peroxide produced during the glucose oxidation activates the wheat germ POD. Ferulic acid oxidation in solutions containing different ratios of POD + GOX or HOX + POD was followed by UV spectrophotometry. For the same dosage, the HOX-POD system is the most efficient for peroxidase activation. Using absorbance data and kinetic constants of GOX and POD, a mathematical model describing the release or consumption of the different reactants (hydrogen peroxide, oxygen, and ferulic acid) in the medium was developed, and experimental data correlated well with calculated values. The results obtained will be applied to investigate the effect of GOX and HOX activities on the rheological properties of dough.     

Effect of Mixing Conditions on the Behavior of Lipoxygenase,Peroxidase, and Catalase in Wheat Flour Doughs

The effect of mixing has been tested on the extractable activities of lipoxygenase, peroxidase, and catalase from dough after 2, 5, and 20 min of mixing, and 30 min of rest period after 20 min of mixing. Different mixing conditions have been studied including temperature, atmosphere, speed, amount of water added to the dough, buffer solutions between pH 3.6 and 7.5 added to the dough, and different additives (linoleic acid, guaiacol, hydrogen peroxide, ascorbic acid, cysteine, yeast, and sodium chloride). In all the mixing conditions tested, the dough peroxidase activity remains equivalent to the initial flour activity, whereas losses in lipoxygenase and catalase activities largely varied according to mixing conditions. The results show that a self-destruction mechanism as well as physicochemical denaturation are responsible for these losses. Lipoxygenase losses seem mainly associated with the former mechanism, whereas catalase losses are highly increased in acidic conditions (physicochemical denaturation). Therefore, the relative impact of the three oxidoreducing enzymes may be largely modulated by mixing conditions.     

Studies on the Glutathione-Dehydroascorbate Oxidoreductase (EC 1.8.5.1) from Wheat Flour

Glutathione (GSH) dehydrogenase was partially purified from wheat flour after extraction, ammonium sulfate precipitation, and ionic-exchange chromatography on diethylaminoethyl (DEAE) Sepharose CL6B. Kinetic studies showed that the optimum pH was close to 7.5. The K_m values varied between 0.15 and 0.28 mM for dehydroascorbic acid (DHA) and between 1.8 and 0.62 mM for GSH when pH was varied from 5.5 to 7.5. The kinetic pattern was consistent with a sequential mechanism for the binding of GSH and DHA. NaCl is a competitive inhibitor with respect to GSH and is uncompetitive with respect to DHA, which suggests that the enzyme combines with DHA before it does with GSH. IsoDHA can replace DHA as hydrogen acceptor but with a K_m of 1.2 mM. γ-Glu-cys was enzymically oxidized but much less efficiently than GSH (V_m = 47 nkat/mL and K_m = 5.5 mM compared to V_m = 362 nkat/mL and K_m = 1.8 mM for GSH), whereas cysteine and cys-gly were not substrates. In the presence of DHA, addition of cysteine and cys-gly to small amounts of GSH causes a large activation of the enzymatic formation of ascorbic acid suggesting coupled oxidation of these thiols.     

Effect of Adding Exogenous Oxidative Enzymes on the Activity of Three Endogenous Oxidoreductases During Mixing of Wheat Flour Dough

The behavior of different exogenous enzymes (soybean lipoxygenase [SLOX], horseradish peroxidase [HPOD], catalase from bovine liver [BCAT], and glucose oxidase [GOX] from Aspergillus niger) added to dough was studied during mixing. The effect of adding these exogenous oxidoreductases on the activity of three oxidative enzymes present in wheat flour (lipoxygenase [WLOX], peroxidase [WPOD], and catalase [WCAT]) was examined. Proper assay conditions were established to differentiate between added WLOX, WPOD, and WCAT and the corresponding activities present in wheat flour. For doughs with added SLOX, an immediate loss of extractable SLOX (≈40%) was observed which remained constant during further mixing. When compared with the control dough, addition of SLOX decreased the losses in WLOX and WCAT activities, whereas WPOD activity was unaffected. With doughs supplemented by HPOD, an immediate loss of 20% in the HPOD activity was observed which did not change after 20 min of mixing. Compared with control dough, addition of HPOD did not affect the behavior of WLOX and WPOD, whereas a slight decrease in the WCAT losses was observed. Addition of BCAT to the dough did not change the behavior of WLOX and WPOD, whereas the losses in WCAT were less rapid. Half of the extractable activity of BCAT was lost at the beginning of mixing with no change during further mixing. For doughs supplemented with GOX, 25% of the GOX activity was lost in the first 5 min of mixing and an additional loss of 20% was observed after 20 min of mixing. Compared with dough without GOX, addition of GOX decreased the losses in WLOX, whereas losses in WCAT and WPOD increased. Glucose and ferulic acid were also added to doughs supplemented with GOX. Added glucose decreased the losses in GOX and WLOX and did not change the behavior of WPOD and WCAT during mixing. Addition of ferulic acid promoted a slight increase of the losses in WLOX and WCAT and almost no change for GOX and WPOD.     

Oxidation of ferulic acid or arabinose-esterified ferulic acid by wheat germ peroxidase

The oxidation of ferulic acid (FA) or 5-O-(trans-feruloyl)-L-arabinose (EFA) by a purified wheat germ peroxidase was followed by UV spectrophotometry and high-performance liquid chromatography using an electrochemical detection. Wheat peroxidase (POD) exhibits a ping-pong bireactant mechanism forming phenoxy radicals more rapidly from FA than from EFA in routine assay conditions. When both the free and the esterified forms of FA are present, the reverse was found. This result could be due to a nonenzymatic cooxidation of FA by the phenoxy radicals of EFA leading to the formation of phenoxy radicals of FA and the EFA regeneration. Addition of ascorbic acid (AA) provokes a delay of FA consumption. AA reduced very rapidly the phenoxy radicals formed by POD back to initial phenol avoiding the formation of ferulate dimers until it was completely oxidized in dehydroascorbic acid. Conversely, cysteine addition slowed but did not delay the FA consumption. The thiol reduced a fraction of the phenoxy radicals produced by wheat POD and was oxidized into cystine, while the other part of phenoxy radicals formed ferulate dimers. These results could be of interest to understand the POD effect on the wheat dough rheological properties.