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Humicola lutea 103 is a copper-tolerant fungal strain able to grow in the presence of 300 μg·mL–1Cu2+under submerged cultivation. To prevent the consequences of copper overload, microorganisms have evolved molecular mechanisms that regulate its uptake, intracellular traffic, storage, and efflux. In spite of this avoidance strategy, high heavy-metal concentrations caused distinct and widespread ultrastructural alterations in H. lutea. The mitochondria were the first and main target of the toxic action. The effect of copper on activities of the key enzymes (hexokinase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, and isocitrate dehydrogenase) included in the 3 main metabolic pathways, glycolysis, pentose phosphate pathway, and tricarboxylic acid cycle, was investigated. High metal concentrations exhibited a dramatic negative effect on hexokinase, while the other 3 enzymes showed a significant and dose-dependent stimulation. On the basis of the present and previous results we concluded that the copper-induced oxidative stress plays an important role in the fungal tolerance to high Cu2+concentrations.

Cu/Zn-containing superoxide dismutase (Cu/Zn-SOD) from the fungal strain Humicola lutea 103 is a naturally glycosylated antioxidant enzyme which has been demonstrated to have a protective effect against myeloid Graffi tumor in hamsters and experimental influenza virus infection in mice. In this work, an improvement of enzyme production was achieved by using both an improved growing strategy and a more efficient purification protocol. The optimized fed-batch cultivation, with 7.5 mg/ml glucose fed daily after 24 h, resulted in prolonged growth and abundant mycelium production, as well as in improvement in enzyme production. A 2-fold increase in the total SOD activity and a significant increase in enzyme production were achieved (from 1.5- to 3.2-fold). Fed-batch technology contributed to a 24 h stabilization period of biosynthesis in which every stop of fermentation can be acceptable, thus making this fermentation a process of industrial interest. In addition, the improved purification procedure offers a reduction of purification steps and enhanced enzyme yield (1.6-fold). The molecular mass was proven to be 15,940 Da for the subunit and the conformational dynamics of the protein in solution was studied by electrospray ionization mass spectrometry (ESI-MS).

Cultures of the fungal strain Humicola lutea 110 were grown in a 3-l bioreactor. Effects of dissolved oxygen concentration (DO) on cell growth, intracellular protein content and antioxidant enzyme activities (SOD and catalase) were investigated. Controlling DO from 20 to 60% lead to: (I). The lethal phase of growth was reached faster; (ii) strong reduction of the intracellular protein content, and (iii) increase of antioxidant enzyme activities. The most efficient SOD biosynthesis was achieved at the 1st maximum of activity in the culture grown under DO uncontrolled conditions.

A novel thermostable MnSOD was purified to electrophoretic homogeneity from the fungal strain Humicola lutea 110. The preparation of the pure metalloenzyme was performed using treatment with acetone followed by ion exchange and gel permeation chromatography. We found that the activity of this enzyme comprises about 80% of the total superoxide dismutase activity in the crude extract, containing two proteins: MnSOD and Cu/ZnSOD. The MnSOD has a molecular mass of approximately 76 kDa and 7200 U/mg protein specific activity. It is a tetrameric enzyme with four identical subunits of 18 860 Da each as indicated by SDS-PAGE, amino acid analysis and mass spectrometry. N-terminal sequence analysis of MnSOD from the fungal strain revealed a high degree of structural homology with enzymes from other eukaryotic sources. Physicochemical properties were determined by absorption spectroscopy and circular dichroism measurements. The UV absorption spectrum was typical for an MnSOD enzyme, but displayed an increased absorption in the 280 nm region (epsilon280 = 10.4 mM(-1). cm(-1)), attributed to aromatic amino acid residues. The CD data show that MnSOD has two negative Cotton effects at 208 and 222 nm allowing the calculation of its helical content. The ellipticity at 222 nm is 6800 deg. x m(2) x dmol(-1) and thus similar to the values reported for other MnSODs. The MnSOD from H. lutea 110 is stable over a wide range of pH (4.5-8), even in the presence of EDTA. The enzyme is thermostable at 70-75 degrees C, and more stable than MnSODs from other sources.

Conidia of Aspergillus niger 26 spores were immobilised in 3% Ca-alginate beads and some intrinsic kinetic characteristics including growth, polymethylgalacturonase (PMG) production and specific oxygen uptake rates determined. A negative correlation between the specific growth rate and specific PMG production indicated a non-growth-associated enzyme formation. Immobilisation did not change the model of PMG synthesis even in repeated replacement cultures and leads under certain conditions to an enhanced PMG production. The immobilised mycelium was concentrated at the surface layer of the alginate beads and the fungal hyphae could not be found in the central part of the gel. The dense growth of the mycelia on the outer bead surface allowed the formation of a fur-like mycelium coat, which had a large surface for oxygen uptake. This growth behaviour is a reason why the effectiveness factor increased during incubation from 0.54 to 0.93.

Pentachlorophenol (PCP) caused a rapid and pronounced increase in the rate of biosynthesis of the superoxide dismutase (SOD) in fungal strain Humicola lutea 110. Mn-containing SOD (Mn-SOD) was mainly responsible for modulating total cell SOD. The kinetics of SOD synthesis in the presence of PCP demonstrated the induction model of enzyme formation. This model was also supported by deinduction experiments, because the removal of the PCP was followed by a marked decrease in SOD activity. PCP also caused a moderate induction of catalase. The concentrations, which were effective in inducing the Mn-SOD, increased the cyanide-resistant respiration. It seems likely that PCP increased the rate of intracellular production of superoxide [Formula: see text]. Addition of inhibitors of transcription and translation to cultures in the presence of inducer (PCP) inhibited further accumulation of SOD activity. These data suggest that PCP, probably by the increase of [Formula: see text] content, accelerates new enzyme synthesis in fungal strain Humicola lutea 110.Key words: superoxide dismutase, superoxide, induction, pentachlorophenol, fungi, Humicola lutea.

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.

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.

The effect of glucose on superoxide dismutase (SOD) activity produced in Humicola lutea 110 was determined. The relatively high glucose concentration in the medium did not repress SOD levels in the cells. Glycerol, a nonfermentable carbon source, caused a slight stimulation of SOD synthesis. Furthermore, the specific rates of enzyme production in the medium with different glucose concentration showed an insignificant difference. Cyclic AMP had no effect on SOD levels. The shift in metabolism as glucose was depleted resulted in an increase in the rate of synthesis of both isocitric dehydrogenase and SOD. Pentachlorophenol and paraquat, which cause the production of superoxide radicals, caused an increase in SOD activity. These results led us to conclude that it is superoxide ion rather than glucose that is controlling SOD levels.Key words: Humicola lutea, fungi, superoxide dismutase, intracellular enzyme, glucose effect.

Spores of fungal strain Humicola lutea 120-5 were immobilized with photo-crosslinkable prepolymer. The entrapped spores were allowed to germinate and develop in situ. The immobilized growing mycelium thus obtained was reused in batch mode for extracellular acid proteinase production. The influence of prepolymer concentration, initial inoculum gel volume, spore content, and duration of the growth cycle on the enzyme activity and mycelium growth were studied. The best yield of the acid proteinase was reached with 15 ml initial gel volume, 20% prepolymer concentration, 10 6 spores per ml gel and 56 h duration of one cycle. The optimization of these factors, which affect the stability and productivity of the immobilized system, resulted in the increase of the enzyme activity (up to 180% compared with free cells), decrease of concentration of outgrowing cells in the medium, and the realization of semicontinuous cultivation. The productive life was nearly 10–12-times longer, and the cumulative enzyme activity was 18-times higher in comparison with those of free culture. Therefore, the immobilized growing Humicola lutea 120−5 in the photo-crosslinked polymer could be used for a long-term production of an acid proteinase.

A novel Cu/Zn-containing superoxide dismutase (SOD) was isolated from the fungal strain Humicola lutea 103. Previously, a protective effect of this enzyme (HLSOD) against tumor growth and also superoxide production in Graffi tumor-bearing hamsters (TBH) were established. The aim of the present study was to investigate the effect of HLSOD on the activity of endogenous SOD and catalase in the cells from TBH during tumor progression. Our results point out that transplantation of Graffi tumor causes a significant decrease in SOD activity in the cells from liver of the hosts (from 35 to 59% compared to the control). In the tumor cells relatively low levels of SOD (about 7 U mg protein-1) were found, and Cu/ZnSOD was the main isoenzyme in total SOD activity. Tumor growth resulted in a reduction of catalase activity, which correlated with the process of tumor progression. A single dose (65 U) treatment with HLSOD caused an increase in endogenous SOD and catalase activity in healthy animals and resulted in restoration of the antioxidant ability in liver cells of the hosts at the early stage of tumor progression. The results show the possible participation of HLSOD in the host oxidant-antioxidant balance, which is probably one of the factors of its immunoprotective action established earlier.

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.

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.