Maria Angelova

Oxidative stress response of filamentous fungi induced by hydrogen peroxide and paraquat.

Although, oxidative stress response, which protects organisms from deleterious effects of reactive oxygen species (ROS), has been extensively studied in pro- and eukaryotes, the information about filamentous fungi is fragmentary. We investigated the effect of two ROS-generating agents (paraquat, PQ, and H2O2) on cellular growth and antioxidant enzyme induction in 12 fungal species. Our results indicate that exposure of fungal spores or mycelia to PQ and H2O2 promoted oxidative stress, as evidenced by remarkable inhibition of spore germination and biomass production; stimulation of cyanide-resistant respiration; accumulation of oxidative modified proteins. Cell responses against both superoxide and peroxide stresses include enhanced expression of superoxide dismutase (SOD) and catalase, however, the extent was different: treatment with PQ increased mainly SOD, whereas exogenous H2O2 led to enhanced catalase. We also found that G6PD has a role in the mechanism of protection against superoxide and peroxide stresses. The activation of antioxidant enzyme defence was blocked by the translation inhibitor, cycloheximide, suggesting that there was de novo enzyme synthesis.

Heat-shock-induced oxidative stress and antioxidant response in Aspergillus niger 26

To extend the knowledge about the relationship between heat shock and oxidative stress in lower eukaryotes, the filamentous fungus Aspergillus niger 26 was chosen as a model system. Here, the response of A. niger cells to heat shock is reported. The temperature treatment significantly increased the levels of reactive oxygen species, superoxide anions (O2), and hydrogen peroxide and the rate of cyanide-resistant respiration as a marker of oxidative stress. Enhanced reactive oxygen species generation coincided with an increase in the content of oxidative damaged protein and in the accumulation of the storage carbohydrates trehalose and glycogen. Thermal survival of the A. niger cells corresponded to a significant increase in the levels of the antioxidant enzymes superoxide dismutase and catalase for all variants. These observations suggest that heat and oxidative stress have a common cellular effect.

A novel glycosylated Cu/Zn-containing superoxide dismutase: production and potential therapeutic effect

The fungal strain Humicola lutea 103 produces a naturally glycosylated Cu/Zn SOD. To improve its yield, the effect of an increased concentration of dissolved oxygen (DO) on growth and enzyme biosynthesis by the producer, cultivated in a 3 l bioreactor, was examined. Exposure to a 20% DO level caused a 1.7-fold increase of SOD activity compared to the DO-uncontrolled culture. Maximum enzyme productivity of SOD was approximately 300 x 10(3) U (kg wet biomass)(-1). The novel enzyme was purified to electrophoretic homogeneity. The presence of Cu and Zn were confirmed by atomic absorption spectrometry. The molecular mass of H. lutea Cu/Zn SOD was calculated to be 31870 Da for the whole molecule and 15936 Da for the structural subunits. The N-terminal sequence revealed a high degree of structural homology with Cu/Zn SOD from other prokaryotic and eukaryotic sources. H. lutea Cu/Zn SOD was used in an in vivo model for the demonstration of its protective effect against myeloid Graffi tumour in hamsters. Comparative studies revealed that the enzyme (i) elongated the latent time for tumour appearance, (ii) inhibited tumour growth in the early stage of tumour progression (73-75% at day 10) and (iii) increased the mean survival time of Graffi-tumour-bearing hamsters. Moreover, the fungal Cu/Zn SOD exhibited a strong protective effect on experimental influenza virus infection in mice. The survival rate increased markedly, the time of survival rose by 5.2 d and the protective index reached 86%. The H. lutea SOD protected mice from mortality more efficiently compared to the selective antiviral drug ribavirin and to commercial bovine SOD. In conclusion, our results suggest that appropriate use of the novel fungal SOD, applied as such or in combination with selective inhibitors, could outline a promising strategy for the treatment of myeloid Graffi tumour and influenza virus infection.

Comparison of antioxidant enzyme biosynthesis by free and immobilized Aspergillus niger cells

Effect of immobilization on antioxidant enzyme synthesis by growing and non-growing cell culture of Aspergillus niger 26 was studied. Entrapped cells showed a greater than 1.5-fold increase in the superoxide dismutase (SOD) activity and a moderate elevation in catalase activity. The immobilization did not cause changes in the spectrum of SOD isoenzymes. The observed increase in SOD activity required de novo synthesis of this enzyme, because it was suppressed by inhibitors of the transcription and translation. The addition of various viscous substances (agar, Na-alginate and pectin) stimulated the SOD synthesis. Despite these results, it was found that the changes in SOD activity are induced in response to growth in the state of immobilization rather than to presence of alginate. Immobilized A. niger cells exhibited about a 4- to 5-fold higher level of cyanide-resistant respiration. This latter phenomenon might use as an indicator of intracellular oxy-intermediate generation in cell culture growing under stress conditions. The results are discussed relative to association between physiological stress caused by immobilization and oxidative stress.

Tuning Genetic Algorithm Parameters to Improve Convergence Time

Fermentation processes by nature are complex, time-varying, and highly nonlinear. As dynamic systems their modeling and further high-quality control are a serious challenge. The conventional optimization methods cannot overcome the fermentation processes peculiarities and do not lead to a satisfying solution. As an alternative, genetic algorithms as a stochastic global optimization method can be applied. For the purpose of parameter identification of a fed-batch cultivation of S. cerevisiae altogether four kinds of simple and four kinds of multipopulation genetic algorithms have been considered. Each of them is characterized with a different sequence of implementation of main genetic operators, namely, selection, crossover, and mutation. The influence of the most important genetic algorithm parameters—generation gap, crossover, and mutation rates has—been investigated too. Among the considered genetic algorithm parameters, generation gap influences most significantly the algorithm convergence time, saving up to 40% of time without affecting the model accuracy.

Role of antioxidant enzymes in survival of conidiospores of Aspergillus niger 26 under conditions of temperature stress

Aims:  A better understanding of the role of antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT) in the protection of Aspergillus niger spores against thermal stress.

Temperature downshift induces antioxidant response in fungi isolated from Antarctica

Although investigators have been studying the cold-shock response in a variety of organisms for the last two decades or more, comparatively little is known about the difference between antioxidant cell response to cold stress in Antarctic and temperate microorganisms. The change of environmental temperature, which is one of the most common stresses, could be crucial for their use in the biotechnological industry and in ecological research. We compared the effect of short-term temperature downshift on antioxidant cell response in Antarctic and temperate fungi belonging to the genus Penicillium. Our study showed that downshift from an optimal temperature to 15° or 6°C led to a cell response typical of oxidative stress: significant reduction of biomass production; increase in the levels of oxidative damaged proteins and accumulation of storage carbohydrates (glycogen and trehalose) in comparison to growth at optimal temperature. Cell response against cold stress includes also increase in the activities of SOD and CAT, which are key enzymes for directly scavenging reactive oxygen species. This response is more species-dependent than dependent on the degree of cold-shock. Antarctic psychrotolerant strain Penicilliumolsonii p14 that is adapted to life in extremely cold conditions demonstrated enhanced tolerance to temperature downshift in comparison with both mesophilic strains (Antarctic Penicilliumwaksmanii m12 and temperate Penicillium sp. t35).

Antioxidant enzyme activity of filamentous fungi isolated from Livingston Island, Maritime Antarctica

From 18 soil samples taken in the vicinity of the permanent Bulgarian Antarctic base “St. Kliment Ohridski” (62°38′29″S, 60°21′53″W) on Livingston Island, 109 filamentous fungi were isolated on selective media. The most widespread fungal species were members of the genera Cladosporium, Geomyces, Penicillium and Aspergillus. Other species, already recorded in Antarctic environment, were also isolated: Lecanicillium muscarium, Epicoccum nigrum and Alternaria alternata. Thirty strains demonstrating good growth were screened for antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) that play an important role in the defense of aerobic organisms against oxidative stress, by converting reactive oxygen species into nontoxic molecules. Six of them showed high enzyme activity. The tested strains produced SOD with statistically significant higher activity at 15°C than at 30°C suggesting that this enzyme is cold-active. Such SOD could be useful in medicine and cosmetics. The best producer of cold-active SOD, Aspergillus glaucus 363, cultivated in bioreactors, demonstrated optimal growth temperature at 25°C and maximum enzyme activities at 25 and 30°C for SOD and CAT, respectively. The electrophoretical analysis showed that the fungus possesses Cu/Zn-SOD.

Effect of cultural conditions on the synthesis of superoxide dismutase by Humicola lutea 110

Abstract Superoxide dismutase (SOD) was produced by the fungal strain Humicola lutea 110. Some of the factors influencing both enzyme and biomass production were studied in shake-flask cultures: glucose and nitrogen concentration, inoculum quantily, and temperature. The best results were obtained for a fermentation performed with 4% glucose, 0.152% nitrogen source, and 8% inoculum at 30°C. The time course of SOD biosynthesis showed two maxima (84.1 and 120.8 U/mg protein SOD activity respectively), which correspond to the maxima of the biomass. The dramatic increase in SOD activity during the late stationary stage indicated that the processes of O2− generation were intensified in the cells. Mn-SOD was responsible for the modulation of total SOD activity at both maxima.

Purposeful model parameters genesis in simple genetic algorithms

Simple genetic algorithms have been investigated aiming to improve the algorithm convergence time. Because of the stochastic nature of genetic algorithms, several runs have to be performed in order to achieve representative results. A procedure for purposeful genesis concerning intervals of variations of model parameters is proposed for a standard simple genetic algorithm, aiming to improve significantly the algorithm effectiveness. Such a stepwise methodology is applied to parameter identification of fed-batch cultivation of S. cerevisiae. The procedure is further validated to a modified simple genetic algorithm with changed sequence of main genetic algorithm operators, namely mutation, crossover and selection, proven to be faster than the standard one. Results obtained from both applications show significant improvement of the algorithm convergence time while saving the model accuracy.