Copper stress and filamentous fungus Humicola lutea 103 — ultrastructural changes and activities of key metabolic enzymes

Humicola lutea 103 is a copper-tolerant fungal strain able to grow in the presence of 300 μg·mL–1 Cu2+ 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 Cu 2+ concentrations.

Examination of Humicola lutea Immobilized in Sol-Gel Matrices: Effective Source of α-Galactosidase

α-Galactosidase production by the fungus Humicola lutea 120-5 immobilized in a hybrid sol-gel matrix, consisting of tetraethylorthosilicate (TEOS) as a precursor and a mixture of polyethyleneglycol (PEG) and polyvinylalcohol (PVA), was investigated under semicontinuous shake flask cultivation and compared to the enzyme secretion by free cells. The influence of the carrier weight on the α-galactosidase biosynthesis in repeated batch experiments was followed. Best results were obtained with 2 g of the sol-gel particles per culture flask using 144-h runs. The growth behaviour of the immobilized mycelium during both the growth and productive phases was observed by scanning electron microscopy. The presence of abundant mycelial growth of intact hyphae correlated with a 2-fold higher enzyme activity compared to free cells. The obtained biocatalyst retained a high level of enzyme titer exceeding the activity of free cells during four cycles of operation (24 days). This result is confirmed by the micrographs showing the retained viability of the growing vegetative cells due to the protective role of the carrier.

Ultracytochemical Localization of Acid Phosphatase in Humicola lutea Conidia and Mycelia

Electron microscopic cytochemical procedures were used to determine the cellular location of acid phosphatase in the fungus Humicola lutea grown in casein-containing medium lacking in mineral orthophosphates. In our investigations acid phosphatase in nongerminating conidia was localized on the outer side of the cell wall, in the cell wall, and on the exterior surface of the plasma membrane. The reaction product of acid phosphatase in germinating conidia was seen in the outer wall layer while in young mycelium on the cell surface and in the exocellular space. The relationship between phosphatase activities localized in the cell wall and their role in the enzymatic degradation of the phosphoprotein casein providing available phosphates for cell growth is discussed.