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.

Bacterial decolorization of acid orange 7 in the presence of ionic and non-ionic surfactants.

The effects of the non-ionic surfactant Triton X-100, the cationic surfactant cetyltri-methylammonium bromide (CTAB) and the anionic surfactant sodium N-lauroyl sarcosinate (SLS) on the decolorization of the reaction medium containing the monoazo dye Acid Orange 7 (AO7) by Alcaligenes faecalis and Rhodococcus erythropolis were studied. It was found that the surfactants influenced in different ways the rate of decolorization. At all concentrations tested the non-ionic surfactant Triton X-100 decreased the decolorization rate of R. erythropolis. At concentrations above the critical micelle concentration (CMC) Triton X-100 upset the usually observed exponential decay of the dye with A. faecalis due probably to the existence of an outer membrane in this organism. In concentrations above the CMC the anionic surfactant SLS inhibited the decolorization and, at prolonged incubation, caused partial release of the bound dye. The cationic surfactant CTAB in concentrations above and below the CMC accelerated drastically the binding of AO7 to the cells causing a rapid staining of the biomass and complete decolorization of the reaction medium. An attempt was made for explanation of the observed differences by the negative electrostatic charge of the living bacterial cell.

Temperature effect on bacterial azo bond reduction kinetics: an Arrhenius plot analysis.

Studied was the effect of temperature in the range 12-46 degrees C on the rate of bacterial decolorization of the mono-azo dye Acid Orange 7 by Alcaligenes faecalis 6132 and Rhodococcus erythropolis 24. With both strains the raise of temperature led to a corresponding raise of decolorization rate better manifested by R. erythropolis. The analysis of the Arrhenius plot revealed a break near the middle of the temperature range. The regression analysis showed practically complete identity of the observed break point temperatures (T (BP)): 20.7 degrees C for Alc. faecalis and 20.8 degrees C for R. erythropolis. The values of the activation energy of the decolorization reaction (E (a)) were found to depend on both the organism and the temperature range. In the range below T (BP) the estimated values of E (a) were 138 +/- 7 kJ mol(-1) for Alc. faecalis and 160 +/- 8 kJ mol(-1) for R. erythropolis. In the range above T (BP) they were 54.2 +/- 1.8 kJ mol(-1) for Alc. faecalis and 37.6 +/- 4.1 kJ mol(-1) for R. erythropolis. Discussed are the possible reasons for the observed abrupt change of the activation energy.

Unusual location and characterization of Cu/Zn-containing superoxide dismutase from filamentous fungus Humicola lutea.

The present study aims to provide new information about the unusual location of Cu/Zn-superoxide dismutase (Cu/Zn-SOD) in lower eukaryotes such as filamentous fungi. Humicola lutea, a high producer of SOD was used as a model system. Subcellular fractions [cytosol, mitochondrial matrix, and intermembrane space (IMS)] were isolated and tested for purity using activity measurements of typical marker enzymes. Evidence, based on electrophoretic mobility, sensitivity to KCN and H(2)O(2) and immunoblot analysis supports the existence of Cu/Zn-SOD in mitochondrial IMS, and the Mn-SOD in the matrix. Enzyme activity is almost equally partitioned between both the compartments, thus suggesting that the intermembrane space could be one of the major sites of exposure to superoxide anion radicals. The mitochondrial Cu/Zn-SOD was purified and compared with the previously published cytosolic enzyme. They have identical molecular mass, cyanide- and H(2)O(2)-sensitivity, N-terminal amino acid sequence, glycosylation sites and carbohydrate composition. The H. lutea mitochondrial Cu/Zn-SOD is the first identified naturally glycosylated enzyme, isolated from IMS. These findings suggest that the same Cu/Zn-SOD exists in both the mitochondrial IMS and cytosol.

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.