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.     

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.     

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.     

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. Correspondence to: B. Tchorbanov     

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.     

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.     

Scanning electron microscopy of immobilizedHumicola lutea during semicontinuous production of acid proteinases

The morphology of the fungusHumicola lutea (strain 120–5), immobilized in polyacrylamide and polyhydroxyethylmethacrylate and used for the semicontinuous production of acid proteinases, was examined by scanning electron microscopy. The fungus developed a dense mycelium below the bead surface as well as in the bead interior after precultivation of entrapped spores. During maximal semicontinuous enzyme biosynthesis, formation of numerous large bulbous cells with a different shape was observed. Lysis of the cells was observed mainly in the centre of the gel beads after 13 successive fermentations with polyacrylamide-immobilized cells or after 21 re-uses of polyhydroxyethylmethacrylate-immobilized mycelia, respectively. Growth and changes in the cellular morphology of immobilizedH. lutea, accompanying biosynthesis of acid proteinases, were comparable in both gel matrices but mycelia immobilized in polyhydroxyethylmethacrylate maintained their productivity twice as long.     

Production of acid proteinase by Humicola lutea 120-5 immobilized in mixed photo-cross-linked polyvinyl alcohol and calcium-alginate beads

Humicola lutea 120-5 spores were immobilized in a mixed photo-crosslinked polyvinyl alcohol and calcium-alginate gel. Maximum enzyme synthesis was established with 1:8 (v:v) gel beads: growth medium inoculum and 48 h duration of one cycle. The free cells were very unstable in replacement fermentations. The operational stability of the immobilized system indicated the possibility of the application of Humicola lutea 120-5 in a semi-continuous process for the production of acid proteinase.     

Extracellular ß-galactosidase production during growth of filamentous fungi on polygalacturonic acid

The ß-galactosidase enzymes of Aspergillus oryzae and Scopulariopsis sp. are secreted during growth of the fungi on polygalacturonic acid, but not during growth on a wide range of other substrates, including lactose. The enzymes are thermotolerant with temperature optima in the range 55–65°C. The results indicate that fungal extracellular ß-galactosidases are involved in fungal growth on complex polysaccharides but not on lactose.     

Extracellular 5-aminolevulinic acid production by Escherichia coli containing the Rhodopseudomonas palustris KUGB306 hemA gene

The Rhodopseudomonas palustris KUGB306 hemA gene codes for 5-aminolevulinic acid (ALA) synthase. This enzyme catalyzes the condensation of glycine and succinyl-CoA to yield ALA in the presence of the cofactor pyridoxal 5'- phosphate. The R. palustris KUGB306 hemA gene in the pGEX-KG vector system was transformed into Escherichia coli BL21. The effects of physiological factors on the extracellular production of ALA by the recombinant E. coli were studied. Terrific Broth (TB) medium resulted in significantly higher cell growth and ALA production than did Luria-Bertani (LB) medium. ALA production was significantly enhanced by the addition of succinate together with glycine in the medium. Maximal ALA production (2.5 g/l) was observed upon the addition of D-glucose as an ALA dehydratase inhibitor in the late-log culture phase. Based on the results obtained from the shake-flask cultures, fermentation was carried out using the recombinant E. coli in TB medium, with the initial addition of 90 mM glycine and 120 mM succinate, and the addition of 45 mM D-glucose in the late-log phase. The extracellular production of ALA was also influenced by the pH of the culture broth. We maintained a pH of 6.5 in the fermenter throughout the culture process, achieving the maximal levels of extracellular ALA production (5.15 g/l, 39.3 mM).     

Conformational and stacking properties of 3′–5′ and 2′–5′ linked oligoribonucleotides studied by CD

Comparative CD studies have been carried out to characterize the properties of 2′–5′ and 3′–5′ oligoriboadenylates and oligoribouridylates from dimer to decamer. The CD band of the 3′–5′ oligoribonucleotides was larger than that of the 2′–5′ oligoribonucleotides and increased with the increase in chain length, while the CD band of the 2′–5′ oligoribonucleotides increased little beyond the dimer level. The CD analysis of the chain length dependency revealed that the 3′–5′ oligoribonucleotides adopt mainly the base-base stacking interaction, while the base-sugar interaction is predominant in the 2′–5′ oligoribonucleotides. The CD intensity of 3′–5′ oligoribonucleotides decreased to a larger extent at elevated temperatures or in the presence of ethanol compared to that of the 2′–5′ counterparts. Mg²⁺ or Mn²⁺ ion enhanced the magnitude of the CD of 3′–5′ octariboadenylate, while a small decrease in the CD was observed by the presence of Mg²⁺ or Mn²⁺ ion to the 2′–5′ octariboadenylate. The 3′–5′ oligoribonucleotide is likely conformationally flexible and can form helical ordered structure with strong base-base stacking depending on changes in the environment such as temperature, the presence of Mg²⁺ ion, or hydrophobicity of the solution. © 1996 John Wiley & Sons, Inc.     

Extracellular enzyme activities during humic acid degradation by the white rot fungi Phanerochaete chrysosporium and Trametes versicolor

Abstract The relationship between humic acid biodegradation and extracellular lignin peroxidase and Mn-dependent peroxidase activities of two white rot fungi, Phanerochaete chrysosporium and Tranetes versicolor , reported to be lignin degraders, was examined. In experimental conditions promoting culture aeration, particularly with T. versicolor no extracellular peroxidase activity could be detected unless humic acids were included in the culture medium. In the presence of humic acids, appreciable enzymatic activities were determined in the culture filtrate of the two fungi. However, T. versicolor was a more effective degrader than P. chrysosporium , and mineralization assays on synthetic humic acids with culture filtrates showed the important role played by Mn²⁺. The surfactant properties of humic acids are suggested to be responsible for the increase of enzymatic activities.     

A calorimetric study of a polymer–monomer complex formed by polyuridylic acid 3′,5′-cyclic AMP

The existence of a soluble complex formed by polyuridylic acid (poly (U)) and 3′,5′-cyclic AMP (cAMP) is demonstrated by u.v. extinction vs. temperature curves, optical rotation, equilibrium dialysis, and reaction calorimetry. The complex hasthe stoichiometry of 2 poly (U)-cAMP and its formation is accompanied by an enthalpy change of ?13.0 kcal/mole of base triplet. The introuction of an empirical factor α in the equations given by Damle² and Crothers² leads to the evolution of a ΔH value of ?13.4 keal/mole. The parameter α is considered as a correction factor for the concentration dependence of the binding process. There is no relation between α and the reduction of monomer activity due to self-association of monomers. The study of the binding process at several temperatures showed that the cooperativity parameter, σ, is independent of temperature and its value of 6.5 × 10^?3 is in good agreement with σ = 5 × 10^?3 for the poly (U)·poly(A) system.³     

Identification of the Pseudornonas aeruginosa acid phosphatase as a phosphorylcholine phosphatase activity

Summary Choline, betaine and N,N-dimethylglycine as the sole carbon and nitrogen source induced a periplasmic acid phosphatase activity in Pseudomonas aeruginosa. This enzyme produced the highest rates of hydrolysis in phosphorylcholine and phosphorylethanolamine among the various phosphoric esters tested. At saturating concentrations of Mg²⁺, the K_m values were 0.2 and 0.7 mM for phosphorylcholine and phosphorylethanolamine respectively. At high concentrations both compounds were inhibitors of the enzyme activity. The K _inf1 ^sups values for phosphorylcholine and phosphorylethanolamine were 1.0 and 3.0 mM respectively. The higher catalytic efficiency was that of phosphorylcholine. Considering these results it is possible to suggest that the Pseudomonas aeruginosa acid phosphatase is a phosphorylcholine phosphatase. The existence of this activity which is induced jointly with phospholipase C by different choline metabolites, in a high phosphate medium, suggests that the attack of Pseudomonas aeruginosa on the cell host may also be produced under conditions of high phosphate concentrations, when the alkaline phosphatase is absent.     

Identification of the adipocyte acid phosphatase as a PAO-sensitive tyrosyl phosphatase.

We have partially purified an 18-kDa cytoplasmic protein from 3T3-L1 cells, which dephosphorylates pNPP and the phosphorylated adipocyte lipid binding protein (ALBP), and have identified it by virtue of kinetic and immunological criteria as an acid phosphatase (EC 3.1.3.2). The cytoplasmic acid phosphatase was inactivated by phenylarsine oxide (PAO) (Kinact = 10 microM), and the inactivation could be reversed by the dithiol, 2,3-dimercaptopropanol (Kreact = 23 microM), but not the monothiol, 2-mercaptoethanol. Cloning of the human adipocyte acid phosphatase revealed that two isoforms exist, termed HAAP alpha and HAAP beta (human adipocyte acid phosphatase), which are distinguished by a 34-amino acid isoform-specific domain. Sequence analysis shows HAAP alpha and HAAP beta share 74% and 90% identity with the bovine liver acid phosphatase, respectively, and 99% identity with both isoenzymes of the human red cell acid phosphatase but no sequence similarity to the protein tyrosine phosphatases (EC 3.1.3.48). HAAP beta has been cloned into Escherichia coli, expressed, and purified as a glutathione S-transferase fusion protein. Recombinant HAAP beta was shown to dephosphorylate pNPP and phosphoALBP and to be inactivated by PAO and inhibited by vanadate (Ki = 17 microM). These results describe the adipocyte acid phosphatase as a cytoplasmic enzyme containing conformationally vicinal cysteine residues with properties that suggest it may dephosphorylate tyrosyl phosphorylated cellular proteins.     

Differentiation of a human monocyte-like cell line by (2′–5′) oligoisoadenylate

Treatment of a human monocyte-like cell line (U-937) by (2'-5')ApApA, the 5' dephosphorylated product of (2'-5')oligo-isoadenylate [oligo(A)] synthetase, an interferon-induced enzyme, was able to induce differentiation, mimicking the effect of interferon treatment. Treatment of U-937 cells with (2'-5')ApApA resulted in morphologic changes, new (monocyte-associated) membrane antigen expression, and acquisition of the capacity to mediate antibody-dependent cellular cytotoxicity (ADCC). (2'-5')ApA and (3'-5')ApApA were without effect. A myeloid cell line (HL-60) which differentiates in response to other agents, but not to alpha-interferon, was not able to differentiate in response to (2'-5')ApApA, despite the ability of interferon to induce (2'-5')oligo (A) synthetase.     

SnTox5–Snn5: a novel Stagonospora nodorum effector–wheat gene interaction and its relationship with the SnToxA–Tsn1 and SnTox3–Snn3–B1 interactions

The Stagonospora nodorum–wheat interaction involves multiple pathogen‐produced necrotrophic effectors that interact directly or indirectly with specific host gene products to induce the disease Stagonospora nodorum blotch (SNB). Here, we used a tetraploid wheat mapping population to identify and characterize a sixth effector–host gene interaction in the wheat–S. nodorum system. Initial characterization of the effector SnTox5 indicated that it is a proteinaceous necrotrophic effector that induces necrosis on host lines harbouring the Snn5 sensitivity gene, which was mapped to the long arm of wheat chromosome 4B. On the basis of ultrafiltration, SnTox5 is probably in the size range 10–30 kDa. Analysis of SNB development in the mapping population indicated that the SnTox5–Snn5 interaction explains 37%–63% of the variation, demonstrating that this interaction plays a significant role in disease development. When the SnTox5–Snn5 and SnToxA–Tsn1 interactions occurred together, the level of SNB was increased significantly. Similar to several other interactions in this system, the SnTox5–Snn5 interaction is light dependent, suggesting that multiple interactions may exploit the same pathways to cause disease.     

Methane production from rice straw pretreated by a mixture of acetic–propionic acid

Rice straw was treated with a mixed solution of acetic acid and propionic acid to enhance its biodegradability. The effect of acid concentration, pretreatment time, and the ratio of solid to liquid on the delignification performance of rice straw were investigated. It was found that the optimal conditions for hydrolysis were 0.75 mol/L acid concentration, 2 h pretreatment time and 1:20 solid to liquid ratio. Batch methane fermentation of untreated rice straw, pretreated rice straw, and the hydrolysates (the liquid fraction) of pretreatment were conducted at 35 °C for 30 days, and the results indicated that methane production of rice straw can be enhanced by dilute organic acid pretreatment. Moreover, most of the acid in hydrolysates can also be converted into methane gas.     

Microbial production of conjugated γ‐linolenic acid from γ‐linolenic acid by Lactobacillus plantarum AKU 1009a

Aims: Optimal production conditions of conjugated γ‐linolenic acid (CGLA) from γ‐linolenic acid using washed cells of Lactobacillus plantarum AKU 1009a as catalysts were investigated. Methods and Results: Washed cells of Lact. plantarum AKU 1009a exhibiting a high level of CGLA productivity were obtained by cultivation in a nutrient medium supplemented with 0·03% (w/v) α‐linolenic acid as an inducer. Under the optimal reaction conditions with 13 mg ml^?1γ‐linolenic acid as a substrate in 5 ‐ml reaction volume, the washed cells [32% (wet cells, w/v) corresponding to 46 mg ml^?1 dry cells] as the catalysts produced 8·8 mg CGLA per millilitre reaction mixture (68% molar yield) in 27 h. The produced CGLA was a mixture of two isomers, i.e., cis‐6,cis‐9,trans‐11‐octadecatrienoic acid (CGLA1, 40% of total CGLA) and cis‐6,trans‐9,trans‐11‐octadecatrienoic acid (CGLA2, 60% of total CGLA), and accounted for 66% of total fatty acid obtained. The CGLA produced was obtained as free fatty acids adsorbed mostly on the surface of the cells of Lact. plantarum AKU1009a. Conclusion: The practical process of CGLA production from γ‐linolenic acid using washed cells of Lact. plantarum AKU 1009a was successfully established. Significance and Impact of the Study: We presented the first example of microbial production of CGLA. CGLA produced by the process is valuable for evaluating their physiological and nutritional effects, and chemical characteristics.