Humicola Lutea Research Articles

Regulation of Superoxide Dismutase Synthesis in Humcola lutea Cells under Cu2+ 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.

Adaptive Response of Humicola Lutea to Copper Exposure

ABSTRACTIn the present study, we examined the adaptive response of the filamentous fungus Humicola lutea 103 to copper stress. Our findings indicate that pretreatment with low concentrations of copper ions led to the development of resistance to high concentrations of the same heavy metal. Spores subjected to 40 μg/ml Cu++ showed higher resistance to killing by subsequent incubation in 70 or 150 μg/ml Cu++ compared to the single treatment. Pre-exposure strategy also caused a higher amount of biomass accumulation and recovery of the fungal growth curves to the control curves shape. Moreover, this strategy exerted protective effect against the oxidative damaged proteins in H. lutea cells, which was accompanied by a corresponding accumulation of reserve carbohydrates such as trehalose and glycogen. Adaptive cell response included induction in the activity of the first antioxidative enzyme superoxide dismutase, but catalase activity was independent of copper treatment and pretreatment. Therefore, we concluded that the pre-exposure to mild copper stress induces a coordinated response that decreases the oxidative stress degree in H. lutea cells when this pre-exposure was followed by severe stress.

Sol-Gel Nanomaterials with Algal Heteropolysaccharide for Immobilization of Microbial Cells, Producing A-Galactosidase and Nitrilase

ABSTRACTThe main purpose of the present work is the sol-gel synthesis and structure of the hybrid nanomaterials as matrices for two types of cells, producing hydrolytic enzymes. The effect of different percent of algal polysaccharide included in them on the hydrolytic activity of fungal and bacterial cells was investigated.The hybrid sol-gel nanomaterials were synthesized from tetraethylortosilicate (TEOS) as a silicon precursor and heteropolysaccharide (AHPS) from the red microalga Dixonella grisea as an organic part. The structure of these matrices was investigated using different methods: FT-IR, XRD, BET-Analysis, EDS, SEM and AFM. The sol-gel hybrids were used for the immobilization of fungal (Humicola lutea) and bacterial (Bacillus sp.) cells, producing α-galactosidase and nitrilase, respectively. It was established the effect of the quantity of the heteropolysaccharide in the matrices on the activity of these hydrolytic enzymes. Using 20% AHPS in the hybrid nanomaterials the α-galactosidase yield exceeded over two-fold the enzyme titre of the free cells in the third cycle of repeated batch shake flask cultivation. These results correlated with a dense growth of immobilized mycelium observed with scanning electron microscopy (SEM). The increase of the percentage of organic part in the sol-gel matrix up to 20% led to an increase in the nitrilase activity. The addition of 40% AHPS did not significantly affect the decrease of the nitrile biodegradation.

Acid phosphatases excreted by Humicola lutea 120-5 in casein-containing medium

The fungus Humicola lutea 120-5 cultivated in casein-containing media, in the presence or absence of inorganic phosphate (Pi), excretes three different molecular forms of acid phosphatase (with Mr values of approximately 140, 70 and 35 kDa). The enzyme forms were isolated and purified 30–100-fold by a procedure involving two steps of ion-exchange chromatography and Sephadex G-200 gel chromatography. It was found that the fungus excretes only one of the phosphatases with the highest Mr (140 kDa) during growth on medium with inorganic nitrogen source (NaNO3). This form (designed AcPh I) was assumed to be a constitutive, since it showed resistance to high Pi-concentrations (10 mM) and its biosynthesis was not affected by the type of nitrogen source (casein or NaNO3). The other two forms (AcPh II-70 and AcPh III-35 kDa) were competitively inhibited by Pi (K i = 0.5 and 0.2 mM, respectively) and were induced by casein. The K m values of AcPh I and AcPh II were estimated as 1.3 mM, while AcPh III showed a higher affinity for p-nitrophenylphosphate (pNPP) with K m of 0.5 mM. The AcPh I–III fractions demonstrated a pH optimum in the range of 4.5–4.8 and an optimal temperature of 55 °C using pNPP as a substrate.

Continuous culture of Humicola lutea 120-5 for acid proteinase production

Acid proteinase production by the fungus Humicola lutea 120-5 in continuous culture was studied. The maximum activity of the culture broth reached 2200 U/ml at a dilution rate (D) of 0.05/h. The continuous process was carried out for 1 month without any bacterial contamination, due to low pH (3.0–3.5) during the cultivation.

Induction of superoxide dismutase synthesis inHumicola lutea110 by pentachlorophenol

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.

Effect of glucose on the superoxide dismutase production in fungal strain Humicola lutea.

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.

Production of Casein Hydrolysates During the Cultivation of Humicola Lutea Cells, Producing Acid Proteinases

ABSTRACTThe casein hydrolysis was studied during the cultivation of Humicola lutea cells producing acid proteinases. The casein from the fermentation medium appeared simultaneously as a nitrogen source, as an inductor for proteinases biosynthesis as well as a substrate for production of casein hydrolysates. Monitoring the cultivation time various casein hydrolysates can be obtained—from partially modified proteins (yield 80%) with improved emulsion properties to peptones (yield 60%) with degree of hydrolysis more than 50%.

Production of casein hydrolysates by extracellular acid proteinases of immobilized Humicola lutea cells

Casein hydrolysis was studied during the cultivation of immobilized Humicola lutea cells producing acid proteinases. By monitoring the cultivation with time, various casein hydrolysates could be obtained, from partially modified proteins (yield 80%) with improved emulsion properties to peptones (yield > 50%) with a degree of hydrolysis >40%. The casein from the fermentation medium appeared to be simultaneously a nitrogen source, an inducer of proteinase biosynthesis, and a substrate for the production of casein hydrolysates. Casein (4%) and glucose (2%) ensured optimal cultivation conditions. The fungal cells, immobilized in calcium alginate beads, required a short cultivation time and demonstrated comparable hydrolysis of casein during five to seven reuses in batch mode.

Production of Acid Proteinases by Humicola Lutea Immobilized in Calcium-Alginate Beads

ABSTRACTSpores of Humicola lutea 120–5 were entrapped in calcium-alginate beads and were cultivated to form an immobilized mycelium used further for acid proteinases production in batch mode. The enzyme yield increased with the repeated cultivations reaching a maximum after eight cycles. The half-life time of enzymes production was achieved by twelve reuses.

Production of acid proteinases by Humicola lutea immobilized in polyacrylamide gel

Fungal spores ofHumicola lutea 120–5 were entrapped in 5% polyacrylamide gel and were cultivated for 44–48 h to form a mycelial network inside the beads. A dense mycelial growth also occurred on the surface of the beads. It was possible to reuse the immobilized mycelium for production of acid proteinases in 12 different batches without loss of mechanical stability. The inoculum size should be controlled prior to its transfer into fresh production medium. Maximal enzyme production exceeding the level of free cell fermentation was registered in the fourth to seventh cycles. According to the size of the inoculum, half of the initial production rate was reached after 7–14 batches.

Selection of gamma-ray mutants from a strain of Humicola lutea 72, producing acid proteases

Gamma-ray exposure was used as a mutagenic agent for mutant induction from a strain of Humicola lutea 72 producing acid proteases. The second stage of irradiation resulted in a double active mutant (H. lutea 120–8). Further treatment gave rise to a triple active mutant (H. lutea 120–5) with a maximal proteolytic activity of 1,800 μg tyrosine liberated from 2% casein/ml−1 culture filtrate/min−1 at pH 3,0 and 40°C. A change in the composition of proteolytic enzymes caused by gamma-ray exposure was established, using chromatography on DEAE-cellulose.