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.     

Acid phosphatase localization in the fungus Whetzelinia sclerotiorum

Acid phosphatase was localized by light and electron microscopy in chains of vacuoles in hyphal tip cells of Whetzelinia sclerotiorum. The Enzyme was present in these vacuoles whether or not conditions favored extracellular acid phosphatase secretion. Apical vesicles, microbodies, woronin bodies, and lipid bodies did not contain acid phosphatase. The implications regarding terminology of organelles in filamentous fungi are discussed with special reference to the fungal spherosome concept.Abbreviations AP acid phosphatase     

Regulation of extracellular acid phosphatase biosynthesis by phosphates in proteinase producing fungus Humicola lutea 120-5

The feasibility of using proteinase producing fungus Humicola lutea 120-5 as a source of extracellular acid phosphatase was investigated. To enhance the acid phosphatase yield and significantly reduce the proteolytic activity the composition of casein-glucose medium containing inorganic phosphate (Pi) was modified. The regulation of phosphatase formation was controlled by Pi. The repression influence of Pi on the synthesis of phosphatase was established. A reduction of Pi (KH(2)PO(4)) concentration from 1.0 to 0.01 g/l caused approximately 5-fold increase of the phosphatase (1200 U/I) and 3-fold decrease of the proteinase (10 U/ml). The omission of Pi from the medium in which the casein (phosphoprotein) was the sole phosphatase source resulted in higher phosphatase yield (2000 U/l) and lower proteolytic activity (7.5 U/ml). Different concentrations of glucose and casein were tested to obtain the optimal medium for maximal acid phosphatase production and minimal level of proteinase. The highest acid phosphatase activity of 2500 U/l and the least amount of acid proteinase (5.5 U/ml) were achieved in 72 h shake-flask culture using Pi-free medium containing glucose and casein in concentrations of 20 and 4 g/l, respectively. The ability of the fungus H. lutea 120-5 to dephosphorylate casein providing orthophosphate for cell growth was discussed.     

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 (P_i), excretes three different molecular forms of acid phosphatase (with M_r 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 M_r (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 P_i-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 P_i (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. This revised version was published online in November 2006 with corrections to the Cover Date.     

Improvement of acid phosphatase production by immobilization of Humicola lutea mycelium in polyurethane sponge

Acid phosphatase production by the fungus Humicola lutea 120-5, immobilized in polyurethane sponge, was studied under semicontinuous shake flask fermentation and compared to the enzyme secretion by free cells. The effect of parameters such as the carrier content and the duration of the batch in repeated batch experiments on the phosphatase production half-life was investigated. The best results were obtained with 1.0 g of sponge cubes (about 1.0 cm per side) per culture flask using 72 h runs. In these conditions the half-life of enzyme production by immobilized biocatalyst was 15 sequential cycles (45 days) compared to three cycles (9 days) for the free mycelium. The maximal phosphatase titre registered in free cell fermentation was 2500 U/l (i.e. 100%), while the relative enzyme activity of the optimal immobilized system was over 100% during the whole half-life time of 45 days. Significant improvement (200–215%) in the yield was observed in one-third of this period or 15 days. The supernatant medium obtained at any stage of the repeated batch cultures did not contain free cells and, due to the low pH (3.0–3.5), the whole process was carried out without any bacterial contamination. In comparison with free cell fermentation, the significant improvement of the acid phosphatase production by polyurethane sponge-immobilized H. lutea mycelium as well as its operation stability was confirmed by scanning electron microscopy.     

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 H₂O₂ 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 H₂O₂-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. Ekaterina Krumova and Alexander Dolashki equally contributed to this work.     

Investigation of acid proteinase biosynthesis by the fungus Humicola lutea 120-5 in an airlift bioreactor

Acid proteinase production using filamentous fungus Humicola lutea 120-5 was studied under batch and continuous fermentation conditions in an airlift bioreactor. A comparison with proteinase production by fungal cells, cultivated in stirred tank bioreactor was made. The process performance in both fermentation devices was similar with respect to substrate utilization, biomass, and enzyme concentration. Continuous acid proteinase production was achieved for 14 days at an optimal dilution rate of 0.05/h with maximum specific activity of 90 U/mg DW of mycelia and yield of 38 U/mg glucose. The volumetric productivity (50 U/ml. h) was approximately 3 times higher than this of the batch system. All continuous experiments were carried out without any bacterial contamination, due to the low pH (3.0-3.5) during the process. The "pellet" type growth of the fungus in the airlift reactor prevented the system from plugging with filaments.     

Acid phosphatase distribution in the liver in cirrhosis

The distribution of acid phosphatase in liver cirrhosis, as well as in its reverse development, was investigated in mice using histochemistry and electron histochemistry methods. Histochemistry demonstrated a sharp activity increase of acid phosphatase (as compared with the same in the material of partial hepatectomy) in liver cells (especially hepatocytes) during liver cirrhosis regression 10 days after a partial hepatectomy. Electron histochemistry has shown the enzyme withdraw out of hepatocytes and connective tissue cells of fibrotic stratum in the extra-cell medium. The reaction product localized on the neighbouring collagen fibres giving evidence that during reverse development of liver cirrhosis the lisosomal enzyme release from specified cells by means of exocytosis and they are involved in the lysis of collagen.     

Extracellular acid phosphatase as a minor enzyme during the production of proteinases by Humicola lutea 120–5

Extracellular acid phosphatase was studied as a minor enzyme of the fungal strain Humicola lutea 120–5 having a clear relation to the secretion of acid proteinases. A medium lacking in mineral orthophosphates ensured a fivefold higher yield of phosphatase while the proteinase production was reduced. An acid phosphatase fraction free of proteinase activity was isolated demonstrating a maximum hydrolysis of 4-nitrophenyl-phosphate at a pH of 4.0 and 50°C. The phosphatase catalyzed a partial dephosphorylation of up to 30% of casein at a pH of 3.0 causing a complete substrate precipitation. Both proteinase and phosphatase biosynthesis increased twofold when natural casein was replaced by partially dephosphorylated casein in the cultivation medium.     

Biomarkers of oxidative stress in the fungal strain Humicola lutea under copper exposure

The fungal strain Humicola lutea 103 was used as a model organism to examine the relationship between copper toxicity and oxidative stress in low eukaryotes such as filamentous fungi. Spores or submerged cultures were treated with different copper concentrations and the oxidative stress-inducing agent paraquat (PQ). Oxidative stress biomarkers such as reactive oxygen species (ROS), cyanide-resistant respiration, protein carbonyls, reserve carbohydrates, and antioxidant defence were identified in cells treated or not treated with either copper ions or PQ. Copper inhibited the growth and conidiospore formation of H. lutea 103 in a concentration-dependent manner. This treatment also resulted in increased superoxide anion radical formation. Copper stress was furthermore accompanied by transient accumulation of trehalose and glycogen, as well as increased protein carbonyl content. Compared to control cultures, copper-treated mycelia demonstrated a marked increase in the activity of protective enzymes (superoxide dismutase, catalase, and glucose-6-phosphate dehydrogenase). These increased antioxidant enzyme activities were blocked by inhibitors of protein synthesis, suggesting that de novo enzyme formation was involved. Biomarker response to the heavy metal was similar to treatment with known ROS generators such as PQ. The observed hyper-oxidative status and increased oxidative damage suggest a relationship between acute metal treatment and oxidative stress in fungal cells.     

Production of acid proteinases by Humicola lutea mycelium immobilized in polyurethane sponge.

Humicola lutea 120-5 spores were entrapped in polyurethane sponge cubes and were cultivated inside the carrier to form an immobilized mycelium further used for production of acid proteinases in batch mode. A carrier—spore suspension ratio of 10:0.5 (wt) should be used to obtain optimal results. The polyurethane sponge-immobilized mycelium could be applied repeatedly, the enzyme activity secreted during the first 10 cycles being about the same as that produced by free cells. The advantages of immobilizing fungal cells by germinating conidia entrapped inside the supporting material are discussed.     

Acid phosphatase localization in the digestive glands of Dionaea muscipula Ellis flytraps

The intracellular localization of acid phosphatases in stimulated digestive glands of Dionaea flytraps has been studied to provide evidence for the route taken by this enzyme during secretion. Previous studies have either included or excluded a role for the dictyosomes in this pathway. Both p-nitrophenyl phosphate and beta-glycerophosphate were used as substrates, and both gave similar localization patterns. Unstimulated glands contained little phosphatase activity in the endomembrane system, whereas 24 and 48 hr after stimulation, heavy deposits of lead were located in the endoplasmic reticulum cisternae, including the nuclear envelope, the dictyosome cisternae, and secretory vesicles. Since dictyosome activation, as judged by the presence of secretory vesicles in the cytoplasm, also coincides with gland stimulation, we conclude that secretion of the hydrolase enzymes occurs via this route and not, as suggested elsewhere, via direct endoplasmic reticulum to plasma membrane connections.     

Purification and properties of trehalase from the thermophilic fungus Humicola lanuginosa.

Trehalase (alpha,alpha-Trehalose glucohydrolase, EC 3.2.1.28) was partially solubilized from the thermophilic fungus Humicola lanuginosa RM-B, and purified 184-fold. The purified enzyme was optimally active at 50 degrees C in acetate buffer at pH 5.5. It was highly specific for alpha,alpha-trehalose and had an apparent Km = 0.4 mM at 50 degrees C. None of the other disaccharides tested either inhibited or activated the enzyme. The molecular weight of the enzyme was around 170 000. Trehalase from mycelium grown at 40 and 50 degrees C had similar properties. The purified enzyme, in contrast to that in the crude-cell free extract, was less stable. At low concentration, purified trehalase was afforded protection against heat-inactivation by "protection against heat-inactivation by "protective factor(s)" present in mycelial extracts. The "protective factor(s)" was sensitive to proteolytic digestion. It was not diffusible and was stable to boiling for at least 30 min. Bovine serum albumin and casein also protected the enzyme from heat-inactivation.     

Acid phosphatase localization in individual neurons by a quantitative histochemical method

—By an adaptation of the fluorometric method of Campbell and Moss (1961), the activity of α-naphthyl acid phosphatase was measured in individual neurons of monkey and human spinal cord and found to be many times higher in nerve cell bodies than in the surrounding neuropil. It was also measured in cerebellar cortex and found most concentrated in the granular (neuronal) layer. As this distribution is distinctive and paralleled by two other acid hydrolases, β-galactosidase and β-glucuronidase, it is considered to offer additional support for the lysosomal concept in nervous tissue and to indicate that nerve cell perikarya are much richer in lysosomes than are axons, dendrites or glial cells.     

Isolation and characterization of a novel superoxide dismutase from fungal strain Humicola lutea 110.

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.     

Structural and functional analysis of glycosylated Cu/Zn-superoxide dismutase from the fungal strain Humicola lutea 103

The fungal strain Humicola lutea 103 produces a naturally glycosylated Cu/Zn-superoxide dismutase (Cu/ZnSOD) (HLSOD). To improve its yield, the effect of increased concentration of Cu2+ (from 1 to 750 microg/ml) on growth and enzyme biosynthesis was studied. The primary structure of this fungal enzyme has been determined by Edman degradation of peptide fragments derived from proteolytic digest. A single chain of the protein, consisting of 152 amino acid residues, reveals a very high degree (74-85%) of structural homology in comparison to the amino acid sequences of other fungal Cu/ZnSODs. The difference of the molecular masses of H. lutea Cu/ZnSOD, measured by MALDI-MS (15,935 Da) and calculated by its amino acid sequence (15,716 Da), is attributed to the carbohydrate chain of one mole of N-acetylglucosamine, attached to the N-glycosylation site Asn23-Glu-Ser. HLSOD protected mice from mortality after experimental influenza A/Aichi/2/68 (H3N2) virus infection. Using the glycosylated HLSOD, the survival rate is increased by 66% (protective index=86.1%) and the survival time prolonged by 5.2 days, similar to the application of ribavarin, while non-glycosylated bovine SOD conferred lower protection.     

Stabilisation of some of the protein synthesis components in the thermophilic fungus,Humicola lanuginosa

The thermal stabilities of tRNA from the thermophilic fungus,Humicola lanuginose were compared with that from the mesophilic yeast,Candida utilis, by measuring the increase in the optical density with temperature. tRNAs from both the species were stable in the presence of millimolar quantities of magnesium chloride upto 50°C, the optimum growth temperature of the fungus. Aminoacyl tRNA synthetases were maximally active at 40°C under thein vitro assay conditions. They were fractionated and one species of valine tRNA synthetase was purified to homogeneity. The purified enzyme was protected against inactivation to varying degrees when preincubated with the substrates valine, tRNA and ATP as well as spermine. Protein turnover studies showed that the rate of turnover was higher at higher temperatures. It was concluded from these results that the protein synthesizing machinery of this fungus has no intrinsic stability but it is stabilised by intracellular factors. Higher rate of protein turnover also plays a role for growth at higher temperature.