Gluconic acid production by Aspergillus niger with oxygen supply by hydrogen peroxide

The production of gluconic acid was carried out with high catalase containing Aspergillus niger mutant. This osmofil strain enables to convert the concentrated solutions of D-glucose (300g/l) to D-gluconic acid without gasing using hydrogen peroxide as oxygen source. A controlled addition of hydrogen peroxide based on the pO₂ measurement was performed. The conversion of 300g/l glucose solution was achieved with 7 hours and triple conversion (with biomass recycling) within 27 hours with yield with regard to the substrate over 98%. Kinetics of inactivation of glucose oxidasecatalase complex as a whole was examined. Some general factors influencing the inactivation of glucose oxidase and catalase in mycelium are discussed.     

The cellular production of hydrogen peroxide

1. The enzyme–substrate complex of yeast cytochrome c peroxidase is used as a sensitive, specific and accurate spectrophotometric H₂O₂ indicator. 2. The cytochrome c peroxidase assay is suitable for use with subcellular fractions from tissue homogenates as well as with pure enzyme systems to measure H₂O₂ generation. 3. Mitochondrial substrates entering the respiratory chain on the substrate side of the antimycin A-sensitive site support the mitochondrial generation of H₂O₂. Succinate, the most effective substrate, yields H₂O₂ at a rate of 0.5nmol/min per mg of protein in state 4. H₂O₂ generation is decreased in the state 4→state 3 transition. 4. In the combined mitochondrial–peroxisomal fraction of rat liver the changes in the mitochondrial generation of H₂O₂ modulated by substrate, ADP and antimycin A are followed by parallel changes in the saturation of the intraperoxisomal catalase intermediate. 5. Peroxisomes supplemented with uric acid generate extraperoxisomal H₂O₂ at a rate (8.6–16.4nmol/min per mg of protein) that corresponds to 42–61% of the rate of uric acid oxidation. Addition of azide increases these H₂O₂ rates by a factor of 1.4–1.7. 6. The concentration of cytosolic uric acid is shown to vary during the isolation of the cellular fractions. 7. Microsomal fractions produce H₂O₂ (up to 1.7nmol/min per mg of protein) at a ratio of 0.71–0.86mol of H₂O₂/mol of NADP⁺ during the oxidation of NADPH. H₂O₂ is also generated (6–25%) during the microsomal oxidation of NADH (0.06–0.025mol of H₂O₂/mol of NAD⁺). 8. Estimation of the rates of production of H₂O₂ under physiological conditions can be made on the basis of the rates with the isolated fractions. The tentative value of 90nmol of H₂O₂/min per g of liver at 22°C serves as a crude approximation to evaluate the biochemical impact of H₂O₂ on cellular metabolism.     

Ammonium-dependent hydrogen peroxide production by mitochondria

NADH-supported generation of H2O2 by permeabilized rat heart mitochondria was partially prevented by the specific complex I-directed inhibitor, NADH-OH, and was significantly stimulated by ammonium. Ammonium did not affect H2O2 production by complex I in coupled submitochondrial particles. The soluble mitochondrial matrix protein fraction catalyzed NADH-dependent H2O2 production, which was greatly (approximately 10-fold) stimulated by ammonium. We conclude that complex I is not the major contributor to mitochondrial superoxide (hydrogen peroxide) generation and that there are specific ammonium-sensitive NADH:oxygen oxidoreductase(s) in the mitochondrial matrix which are responsible for mitochondrial H2O2 production.     

Mitochondrial hydrogen peroxide production alters oxygen consumption in an oxygen-concentration-dependent manner

Metabolic responses of mammalian cells toward declining oxygen concentration are generally thought to occur when oxygen limits mitochondrial ATP production. However, at oxygen concentrations markedly above those limiting to mitochondria, several mammalian cell types display reduced rates of oxygen consumption without energy stress or compensatory increases in glycolytic ATP production. We used mammalian Jurkat T cells as a model system to identify mechanisms responsible for these changes in metabolic rate. Oxygen consumption was 31% greater at high oxygen (150–200 μM) compared to low oxygen (5–10 μM). Hydrogen peroxide was implicated in the response as catalase prevented the increase in oxygen consumption normally associated with high oxygen. Cell-derived hydrogen peroxide, predominately from the mitochondria, was elevated with high oxygen. Oxygen consumption related to intracellular calcium turnover was shown, through EDTA chelation and dantrolene antagonism of the ryanodine receptor, to account for 70% of the response. Oligomycin inhibition of oxygen consumption indicated that mitochondrial proton leak was also sensitive to changes in oxygen concentration. Our results point toward a mechanism in which changes in oxygen concentration influence the rate of hydrogen peroxide production by mitochondria, which, in turn, alters cellular ATP use associated with intracellular calcium turnover and energy wastage through mitochondrial proton leak.     

The production of hydroxyl radical from hydrogen peroxide

The formation of hydroxyl radical (OH·) from the oxidation of glutathione, ascorbic acid, NADPH, hydroquinone, catechol, and riboflavin by hydrogen peroxide was studied using a range of enzymes and copper and iron complexes as possible catalysts. Copper-1,10-phenanthroline appears to catalyze the production of OH· from hydrogen peroxide without superoxide radical being formed as an intermediate, and without the involvement of a catalyzed Haber-Weiss (Fenton) reaction. Superoxide radical is involved, however, in the Cu²⁺ -catalyzed decomposition of hydrogen peroxide, and in the oxidation of glutathione by atmospheric oxygen. For this latter oxidation, copper-4,7-dimethyl-1,10-phenanthroline was found to be a much more effective catalyst than the copper complex of 1,10-phenanthroline, which is normally used. Mechanisms for these reactions are proposed, and the toxicological significance of the ability of a variety of biological reductants to provide a prolific source of OH· when oxidized by hydrogen peroxide is discussed.     

Superoxide and hydrogen peroxide production by Drosophila mitochondria

Drosophila melanogaster is a key model organism for genetic investigation of the role of free radicals in aging, but biochemical understanding is lacking. Superoxide production by Drosophila mitochondria was measured fluorometrically as hydrogen peroxide, using its dependence on substrates, inhibitors, and added superoxide dismutase to determine sites of production and their topology. Glycerol 3-phosphate dehydrogenase and center o of complex III in the presence of antimycin had the greatest maximum capacities to generate superoxide on the cytosolic side of the inner membrane. Complex I had significant capacity on the matrix side. Center i of complex III, cytochrome c, and complex IV produced no superoxide. Native superoxide generation by isolated mitochondria was also measured without added inhibitors. There was a high rate of superoxide production with sn-glycerol 3-phosphate as substrate; two-thirds mostly from glycerol 3-phosphate dehydrogenase on the cytosolic side and one-third on the matrix side from complex I following reverse electron transport. There was little superoxide production from any site with NADH-linked substrate. Superoxide production by complex I following reverse electron flow from glycerol 3-phosphate was particularly sensitive to membrane potential, decreasing 70% when potential decreased 10 mV, showing that mild uncoupling lowers superoxide production in the matrix very effectively.     

Glycerophosphate-dependent hydrogen peroxide production by rat liver mitochondria

We studied the extent to which hormonally-induced mitochondrial glycerophosphate dehydrogenase (mGPDH) activity contributes to the supply of reducing equivalents to the mitochondrial respiratory chain in the rat liver. The activity of glycerophosphate oxidase was compared with those of NADH oxidase and/or succinate oxidase. It was found that triiodothyronine-activated mGPDH represents almost the same capacity for the saturation of the respiratory chain as Complex II. Furthermore, the increase of mGPDH activity induced by triiodothyronine correlated with an increase of capacity for glycerophosphate-dependent hydrogen peroxide production. As a result of hormonal treatment, a 3-fold increase in glycerophosphate-dependent hydrogen peroxide production by liver mitochondria was detected by polarographic and luminometric measurements.     

Aeration without air: oxygen supply by hydrogen peroxide.

Oxygen has been supplied to suspensions of microorganisms kept under nitrogen by the addition of hydrogen peroxide. If catalase was present in the suspension and the flow was adjusted to the rate of oxygen consumption, the cells grew at rates identical to the controls incubated under air. The applicability of oxygen supply by hydrogen peroxide and its limits are discussed.     

Hydroxyl radical and singlet oxygen production and DNA damage induced by carcinogenic metal compounds and hydrogen peroxide

Carcinogenic chromium(VI), iron(III) nitrilotriacetate, cobalt(II), and nickel(II) react with hydrogen peroxide leading to the production of active species including hydroxyl radical and singlet oxygen, which cause DNA damage.     

氧对口腔链球菌产生过氧化氢的影响

目的观察环境中氧含量对口腔链球菌过氧化氢产生速率的影响。方法采用ABTS-HRP微量板法测定在不同氧含量条件下口腔链球菌过氧化氢产生的速率。结果口腔链球菌在严格厌氧条件下过氧化氢产生速率为9.29nmol/(min×109细胞);当氧含量增高,口腔链球菌过氧化氢合成速率加快;在不同氧含量的环境中口腔链球菌产生过氧化氢的速率差异存在显著性:有氧振荡培养>有氧静置培养>厌氧(P<0.05)。结论环境中氧含量是影响口腔链球菌过氧化氢产生速率的重要因素。     

Regulation of hydrogen peroxide production by brain mitochondria by calcium and Bax

Abnormal accumulation of Ca2+ and exposure to pro-apoptotic proteins, such as Bax, is believed to stimulate mitochondrial generation of reactive oxygen species (ROS) and contribute to neural cell death during acute ischemic and traumatic brain injury, and in neurodegenerative diseases, e.g. Parkinson's disease. However, the mechanism by which Ca2+ or apoptotic proteins stimulate mitochondrial ROS production is unclear. We used a sensitive fluorescent probe to compare the effects of Ca2+ on H2O2 emission by isolated rat brain mitochondria in the presence of physiological concentrations of ATP and Mg2+ and different respiratory substrates. In the absence of respiratory chain inhibitors, Ca2+ suppressed H2O2 generation and reduced the membrane potential of mitochondria oxidizing succinate, or glutamate plus malate. In the presence of the respiratory chain Complex I inhibitor rotenone, accumulation of Ca2+ stimulated H2O2 production by mitochondria oxidizing succinate, and this stimulation was associated with release of mitochondrial cytochrome c. In the presence of glutamate plus malate, or succinate, cytochrome c release and H2O2 formation were stimulated by human recombinant full-length Bax in the presence of a BH3 cell death domain peptide. These results indicate that in the presence of ATP and Mg2+, Ca2+ accumulation either inhibits or stimulates mitochondrial H2O2 production, depending on the respiratory substrate and the effect of Ca2+ on the mitochondrial membrane potential. Bax plus a BH3 domain peptide stimulate H2O2 production by brain mitochondria due to release of cytochrome c and this stimulation is insensitive to changes in membrane potential.     

Superoxide and hydrogen peroxide in oxygen damage.

Escherichia coli were damaged and killed by exposure to hyperbaric oxygen. Lethality was measured as the decrease in the number of colonies formed upon plating the exposed cells onto rich agar. Damage was assessed by plating onto both rich and minimal agar. Cells which gave rise to visible colonies on rich but not on minimal agar were considered to be damaged. That this differential colony count was largely due to reparable damage rather than to stable mutagenesis was shown by replica plating from the rich onto the minimal agar. Most of the cells which had been unable to grow when directly plated onto minimal agar regained this ability after growth upon rich agar. Repair of the damage imposed by exposure to oxygen was thus more readily accomplished on a nutritionally rich medium. The enzymes superoxide dismutase, catalase, and peroxidase appeared to protect against oxygen damage. It is thus likely that both O₂^? and H₂O₂ are important agents of oxygen toxicity. In accord with this conclusion were the observations that augmented intracellular levels of these enzymes correlated with increased resistance towards oxygen damage, whereas increased respiratory capacity correlated with increased sensitivity towards hyperbaric oxygen.     

Robust oxygen supply by controlled addition of hydrogen peroxide to microbial cultures

The supply of oxygen can be improved by the direct addition of hydrogen peroxide to cultures of aerobic microbes expressing sufficient amounts of catalase. This is of special interest if normal aeration has to be kept low, for instance, in order to minimize evaporation of volatile compounds (either substrates or products) or to minimize foaming. Also, if the mechanical power input to the bioreactor is or has to be limited, addition of hydrogen peroxide may be useful. The appropriate dosage of hydrogen peroxide can be simply determined by a controller of the oxygen partial pressure or of the oxygen content in the exhaust gas using various control algorithms. The added hydrogen peroxide can be either a stabilized concentrate, e.g. 30%, or any dilute form of this. In high density cultures, Pseudomonas cells tolerated even harsh controller disturbances. This approach proved to be very robust and reliable.