Radoslav Abrashev

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.

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.

ROSMARINIC ACID AND ANTIOXIDANT ENZYME ACTIVITIES IN LAVANDULA VERA MM CELL SUSPENSION CULTURE: A COMPARATIVE STUDY

The growth and intracellular protein content of lavender (Lavandula vera MM) cell suspension culture was followed along with some antioxidant defense system members—non-enzymatic (rosmarinic acid) and enzymatic [superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6)]. It was found that the media content and the cultivation mode strongly influenced the production of plant defense compounds as well as the ratio between non-enzymatic and enzymatic ones. The bioreactor culture contains about two times more rosmarinic acid, superoxide dismutase, and catalase compared to the shake-flask cultivation. These findings are discussed with respect to the relative stress levels and plant antioxidant orchestra system. It was concluded that investigated defense system components (enzymatic and non-enzymatic) were closely associated in a complex balance. The three isoenzyme forms of SOD (Cu/ZnSOD, FeSOD, and MnSOD) in the cells of Lavandula vera were revealed by polyacrylamide gel electrophoresis analysis, and the FeSOD isoform exhibited highest activity.

Oxidative stress-associated impairment of glucose and ammonia metabolism in the filamentous fungus, Aspergillus niger B1-D

Oxidative stress events have been shown to be associated with reduced consumption of nutrients in yeasts, but there are very few studies in filamentous fungi. In the present study we investigated the impact of oxidative stress on glucose and ammonia utilization in batch cultures of Aspergillus niger B1-D. The addition of 1mM H(2)O(2) significantly reduced both glucose and ammonia uptake rates in these cultures. Associated with the decreased nutrient uptake, the activity of glyceraldehyde-3-phosphate dehydrogenase was greatly reduced; conversely, the activity of glucose-6-phosphate dehydrogenase remained unchanged. During the period of reduced nutrient uptake, the intracellular ATP and NADPH levels decreased while the amount of trehalose increased. The activities of glutamine synthetase and glutamate dehydrogenase, two key enzymes of ammonia assimilation, remained unchanged in response to H(2)O(2) up to 1mM, suggesting the decreased ammonia uptake rate noted under such conditions is not due to enzyme inactivation caused by oxidative stress, but may be due to an insufficient supply of ATP and NADPH, which are required for ammonia assimilation.

Biochemical properties of Cu/Zn-superoxide dismutase from fungal strain Aspergillus niger 26

The fungal strain Aspergillus niger produces two superoxide dismutases, Cu/Zn-SOD and Mn-SOD. The primary structure of the Cu/Zn-SOD has been determined by Edman degradation of peptide fragments derived from proteolytic digests. A single chain of the protein, consisting of 153 amino acid residues, reveals a very high degree of structural homology with the amino acid sequences of other Aspergillus Cu/Zn-SODs. The molecular mass of ANSOD, measured by MALDI-MS and ESI-MS, and calculated by its amino acid sequence, was determined to be 15821 Da. Only one Trp residue, at position 32, and one disulfide bridge were identified. However, neither a Tyr residue nor a carbohydrate chain occupying an N-linkage site (-Asn-Ile-Thr-) were found. Studies on the temperature and pH dependence of fluorescence, and on the temperature dependence of CD spectroscopic properties, confirmed that the enzyme is very stable, which can be explained by the stabilising effect of the disulfide bridge. The enzyme retains about 53% of its activity after incubation for a period of 30 min at 60 degrees C, and 15% at 85 degrees C.

Differential Effect of Paraquat and Hydrogen Peroxide on the Oxidative Stress Response in Vibrio Cholerae Non O1 26/06

ABSTRACT Vibrio cholerae non O1 26/06, a non-pathogenic strain, was subjected to treatment by different concentration of paraquat (PQ) and H2O2. Exposure to PQ for 1 h caused induction of reactive oxygen species (ROS) such as superoxide anion radical (•O2−) and H2O2. At the same time, second stress factor significantly inhibited •O2− production and enhanced the intracellular H2O2 content. The enhanced ROS generation resulted in a significant increase in the levels of oxidatively damaged proteins in comparison to the control variant. Thus, the exposure of V. cholerae cells to PQ and H2O2 promoted oxidative stress. Cell response against this stress includes activation of antioxidant enzyme defence. The treatment with PQ concentrations in the range of 0 - 5 mM resulted mainly in activation of SOD, but not noticeably changed CAT activity in V. cholerae non-O1 26/06. In contrast, effect of H2O2treatment on antioxidant enzyme synthesis in our Vibrio strain was still much more pronounced for CAT than for SOD. Therefore, oxidative stress responses induced by •O2− (generated intracellularly by PQ) and H2O2 demonstrated differential adaptation of Vibrio cells to different toxic agents.

Superoxide Dismutase and Catalase Activities in Vibrio Cholerae Non-O1 Strains

ABSTRACT Antioxidant enzymes are essential for living cells, producing protection from reactive oxygen species such as superoxide, which cause oxidative damage to cell structures. Superoxide dismutase (SOD) and catalase (CAT) activities were determined for three Vibrio strains (Vibrio cholerae non O1/29, V. cholerae non O1/29-T and V. cholerae non O1/26) aerobically grown at 30°C (optimal level) and 10 °C (cold stress). All strains tested expressed both antioxidant enzymes under normophysiological and stress conditions, but the cell response is more strain-dependent than dependent on temperature. Levels of SOD in cultures of V. cholerae non O1/26 grown at 30°C were about 2.5 to 9.5-times higher than those in the cultures grown at 10°C. In contrast, SOD activity in V. cholerae non O1/29 increased by 7.5-fold under stress conditions in comparison to that at optimal temperature. The strain V. cholerae non O1/29-T did not show any significant difference in the cell response depending on the growth temperature. CAT activity in cells of V. cholerae non O1/26 and V. cholerae non O1/29-T exhibited a similar tendency suggesting that this enzyme is not included in antioxidant response against cold stress. Contrary to the above observations, V. cholerae non 01/29 demonstrated higher CAT activity in response to temperature downshift. Only one SOD isoenzyme was detected in each of the three Vibrio strains by native PAGE analysis.

Potential of ligninolytic enzymatic complex produced by white-rot fungi from genus Trametes isolated from Bulgarian forest soil

Because of the crucial role of ligninolytic enzymes in a variety of industrial processes, the demand for a new effective producer has been constantly increasing. Furthermore, information on enzyme synthesis by autochthonous fungal strains is very seldom found. Two fungal strains producing ligninolytic enzymes were isolated from Bulgarian forest soil. They were identified as being Trametes trogii and T. hirsuta. These two strains were assessed for their enzyme activities, laccase (Lac), lignin peroxidase (LiP) and Mn‐dependent peroxidase (MnP) in culture filtrate depending on the temperature and the type of nutrient medium. T. trogii was selected as the better producer of ligninolytic enzymes. The production process was further improved by optimizing a number of parameters such as incubation time, type of cultivation, volume ratio of medium/air, inoculum size and the addition of inducers. The maximum activities of enzymes synthesized by T. trogii was detected as 11100 U/L for Lac, 2.5 U/L for LiP and 4.5 U/L for MnP after 14 days of incubation at 25°C under static conditions, volume ratio of medium/air 1:6, and 3 plugs as inoculum. Among the supplements tested, 5% glycerol increased Lac activity to a significant extent. The addition of 1% veratryl alcohol had a positive effect on MnP.

REGULATION OF SUPEROXIDE DISMUTASE SYNTHESIS IN HUMICOLA LUTEA CELLS UNDER Cu 2+ STRESS CONDITIONS

The superoxide dismutase (SOD) is the first enzyme to respond against oxygen radicals and to offer the greatest response to oxidative stress. The great interest in SODs is a result of their important physiological role and therapeutic potential. The fungal strain Humicola lutea 103 has been selected as an effective producer of Cu/Zn-SOD exhibiting protective effect against influenza virus infection and myeloid Graffi tumour. The present paper s the role of control mechanisms induction and catabolite repression on SOD synthesis under copper stress conditions. Cu ions cause rapid and profound induction of enzyme activity by dose- and time-dependent manner. The deinduction experiments also demonstrate the induction model of SOD synthesis. The results suggest that SOD synthesis in the fungal strain H. lutea 103 treated with Cu ions is a subject to catabolite repression.