Production and characterization of human granulocyte–macrophage colony-stimulating factor (hGM-CSF) expressed in the oleaginous yeast Yarrowia lipolytica

Since its isolation, the human granulocyte-macrophage colony-stimulating factor (hGM-CSF) has been proposed as a new class of therapeutic biological products in the treatment of various diseases. However, the toxicity of this cytokine towards its expression host constitutes a major obstacle to bioprocess development for large-scale production. In this work, the optimized gene encoding hGM-CSF was expressed in the yeast Yarrowia lipolytica in one and two copies under the control of the fatty acid-inducible POX2 promoter. Protein secretion was directed by the targeting sequence of the extracellular lipase (LIP2): preXALip2. After 48?h of induction, Western blot analysis revealed the presence of a nonglycosylated form of 14.5?kDa and a trail of hGM-CSF hyperglycosylated varying from 23?kDa to more than 60?kDa. The two-copy transformants produced hGM-CSF level which was sevenfold higher compared to the single-copy ones. Deglycosylation with PNGase F showed two forms: a mature form of 14.5?kDa and an unprocessed form of 18?kDa. The addition of two alanines to the signal sequence resulted in correct hGM-CSF processing. The production level was estimated at 250?mg/l after preliminary optimization studies of the cultivation and induction phases. The purified hGM-CSF was identified by N-terminal sequencing and LC-MS/MS analysis; its biological activity was confirmed by stimulating the proliferation of TF1 cell line. This study demonstrated that Y. lipolytica is a promising host for the efficient production of active toxic proteins like hGM-CSF.     

Toxicities effects of pharmaceutical, olive mill and textile wastewaters before and after degradation by Pseudomonas putida mt-2

Background  

Removal of numerous classes of chemical pollutants from the industrial wastewater such as textile, pharmaceutical and olive mill using conventional wastewater treatment, is incomplete and several studies suggested that improvement of this situation would require the application of biological treatment techniques. Dyes, polyphenols and drugs are an environmental pollutants extremely toxics to plants and other living organisms including humans. These effluents were previously treated by Pseudomonas putida. The main of this work was to evaluate the in vivo toxicity of the three wastewaters.     

Designing universal primers for the isolation of DNA sequences encoding Proanthocyanidins biosynthetic enzymes in Crataegus aronia

ABSTRACT: BACKGROUND: Hawthorn is the common name of all plant species in the genus Crataegus, which belongs to the Rosaceae family. Crataegus are considered useful medicinal plants because of their high content of proanthocyanidins (PAs) and other related compounds. To improve PAs production in Crataegus tissues, the sequences of genes encoding PAs biosynthetic enzymes are required. FINDINGS: Different bioinformatics tools, including BLAST, multiple sequence alignment and alignment PCR analysis were used to design primers suitable for the amplification of DNA fragments from 10 candidate genes encoding enzymes involved in PAs biosynthesis in C. aronia. DNA sequencing results proved the utility of the designed primers. The primers were used successfully to amplify DNA fragments of different PAs biosynthesis genes in different Rosaceae plants. CONCLUSION: To the best of our knowledge, this is the first use of the alignment PCR approach to isolate DNA sequences encoding PAs biosynthetic enzymes in Rosaceae plants.     

Dissolved nitrogen in drinking water resources in Al-Mahareth village of Assir – Saudi Arabia

A water quality study was carried out on ground water wells, which serve as drinking water sources in farming communities in Al-Mahareth village of Assir region of the Kingdom of Saudi Arabia. The objective of this research was to determine concentrations of different forms of nitrogen in drinking water samples. Water samples were collected from these sources every 3 months (from January to December 2008) and analyzed for ammonia, nitrate and nitrite using the Plaintest Photometer Method. Results indicated that the annual mean concentration of nitrate, nitrite and ammonia varied from 23.09 to 25.06 mg/l, 0.006 to 0.36 mg/l and 0.008 to 0.179 mg/l, respectively. An important observation was that, in general, higher nitrate and nitrite concentrations were found during the rainy season compared to the dry season. Concentrations of these potentially toxic substances were below WHO acceptable limits for surface and ground waters, indicating that these water resources appear safe for drinking from a dissolved nitrogen perspective.     

Use of cheese whey to enhance <Emphasis Type="Italic">Geotrichum candidum</Emphasis> biomass production in olive mill wastewater

Geotrichum candidum is a yeast-like filamentous fungus that has attracted industrial interest. The present work investigated G. candidum biomass production in agro-industrial wastewaters (olive mill wastewater (OMW) and cheese whey (CW)) as the only substrate. Different solid media (Sabouraud dextrose agar (SDA), CW, OMW, and OMW/CW mixtures in different proportions) were tested. OMW/CW mixtures proved to be suitable for optimal mycelia growth of G. candidum with a very high hyphae density. The highest fungal and expansion rate growth of 83 ± 1 mm and 12.4 day⁻¹, respectively, were obtained on a 20:80 mixture of OMW/CW, which was incubated for 7 days. This optimal mixture was used to study the biomass production and the OMW decolorization ability of G. candidum in the presence of CW in liquid medium. Liquid cultures were also conducted in OMW and CW separately. After 5 days of incubation, fungal biomass reached 9.26 g l⁻¹ in the OMW/CW mixture and only 2.83 g l⁻¹ in CW, while no biomass production was observed in OMW alone. OMW decolorization and dephenolization by G. candidum also improved in the presence of CW with a decolorization efficiency of 54.5% and a total phenolic reduction of 55.3%, compared with the control which yielded values of about 10% and 15%, respectively. These results suggested that OMW/CW—as the only substrate—could be used as a cost-effective medium to produce G. candidum biomass, without the need for water dilution or supplementation with other nutriments.     

Culture conditions of tannase production by Lactobacillus plantarum

Lactobacillus plantarum produced an extracellular tannase after 24 h growth on minimal medium of amino acids containing 2 g tannic acid l^–1. Enzyme production (6 U ml^–1) was optimal at 37 °C and pH 6 with 2 g glucose l^–1 and 7 g tannic acid l^–1 in absence of O₂.     

Comparative analysis of folding and substrate binding sites between regulated hexameric type II citrate synthases and unregulated dimeric type I enzymes.

We describe the first structure determination of a type II citrate synthase, an enzyme uniquely found in Gram-negative bacteria. Such enzymes are hexameric and are strongly and specifically inhibited by NADH through an allosteric mechanism. This is in contrast to the widespread dimeric type I citrate synthases found in other organisms, which do not show allosteric properties. Our structure of the hexameric type II citrate synthase from Escherichia coli is composed of three identical dimer units arranged about a central 3-fold axis. The interactions that lead to hexamer formation are concentrated in a relatively small region composed of helix F, FG and IJ helical turns, and a seven-residue loop between helices J and K. This latter loop is present only in type II citrate synthase sequences. Running through the middle of the hexamer complex, and along the 3-fold axis relating dimer units, is a remarkable pore lined with 18 cationic residues and an associated hydrogen-bonded network. Also unexpected was the observation of a novel N-terminal domain, formed by the collective interactions of the first 52 residues from the two subunits of each dimer. The domain formed is rich in beta-sheet structure and has no counterpart in previous structural studies of type I citrate synthases. This domain is located well away from the dimer-dimer contacts that form the hexamer, and it is not involved in hexamer formation. Another surprising observation from the structure of type II E. coli citrate synthase is the unusual polypeptide chain folding found at the putative acetylcoenzyme A binding site. Key parts of this region, including His264 and a portion of polypeptide chain known from type I structures to form an adenine binding loop (residues 299-303), are shifted by as much as 10 A from where they must be for substrate binding and catalysis to occur. Furthermore, the adjacent polypeptide chain composed of residues 267-297 is extremely mobile in our structure. Thus, acetylcoenzyme A binding to type II E. coli citrate synthase would require substantial structural shifts and a concerted refolding of the polypeptide chain to form an appropriate binding subsite. We propose that this essential rearrangement of the acetylcoenzyme A binding part of the active site is also a major feature of allostery in type II citrate synthases. Overall, this study suggests that the evolutionary development of hexameric association, the elaboration of a novel N-terminal domain, introduction of a NADH binding site, and the need to refold a key substrate binding site are all elements that have been developed to allow for the allosteric control of catalysis in the type II citrate synthases.     

Incidence and seroprevalence of canine ehrlichiosis in the Medjez El Bab region (northwestern Tunisia during 1994, 1995 and 1996

A sero-epidemiological survey, realized in the Medjez El Bab region (North-West of Tunisia), has concerned 180 dogs which status has been determined during the study. The animals were identified, then underwent an annual blood sampling during three successive years, in order to search for antibodies against E. canis and E. chaffeensis by indirect immunofluorescence. The results show that, in all sero-positive dogs, the levels of antibodies against E. canis were higher than those against E. chaffeensis. The sero-prevalence of E. canis was 42.8%, 50% and 48.9%, in 1994, 1995 and 1996, respectively, and was higher than that against E. chaffeensis during the three year studies. The incidence of E. canis infection was 12.6% during the three years whereas E. chaffeensis infection did not exceed 4.7%.