Aspergillus terreus produces itaconic acid at low pH but lovastatin and other secondary metabolites at higher pH in the fermentation. The utilization of glucose as a carbon substrate was investigated ...for secondary metabolite production by A. terreus. With a starting pH of 6·5, glucose was rapidly metabolized to gluconic acid by the wild-type strain and by transformants harbouring Aspergillus niger genes encoding 6-phosphofructo-1-kinases with superior kinetic and regulatory properties for bioproduction of metabolites from glucose. On exhaustion of the glucose in batch fermentations, the accumulated gluconic acid was utilized as a carbon source. A novel pathway of glucose catabolism was demonstrated in A. terreus, a species whose wild type is, without any strain development, capable of producing gluconic acid at high molar conversion efficiency (up to 0·7 mol mol⁻¹ glucose consumed). Aspergillus terreus is a potential novel producer organism for gluconic acid, a compound with many uses as a bulk chemical. With a new knowledge of glucose catabolism by A. terreus, fermentation strategies for secondary metabolite production can be devised with glucose feeding using feedback regulation by pH.
Cellular nanovesicles (CNVs), that are shed from cells, have been recognized as promising indicators of health status. We analyzed the effect of long-distance running on concentration of CNVs, along ...with some standard blood parameters, in 27 athletes two days before and >15 hours after physical effort.
CNVs were isolated by repetitive centrifugation and washing of samples, and assessed by flow cytometry. Cholinesterase (ChE) and glutathione S-transferase (GST) activity were measured spectrophotometrically. Interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) concentrations were measured using enzyme-linked immunosorbent assay (ELISA). C-reactive protein (CRP) was measured with immunoturbidimetric determination and lipidogram parameters were measured by enzymatic colorimetric assay. Flow cytometry was used for blood cell count and mean platelet volume (MPV) measurement.
More than 15 hours after physical effort a decrease was found in CNVs' concentration in isolates from blood (46%; p<0.05), in ChE activity in whole blood (47%; p<0.001), in plasma (34%; p<0.01), and in erythrocyte suspension (54%; p<0.001), as well as in GST activity in erythrocyte suspension (16%; p<0.01) and in IL-6 concentration in plasma (63%; p<0.05). We found no change in GST activity in plasma and in TNF-α concentration in plasma. Correlations (>0.8; p<0.001) between CNVs' concentration and ChE activity, and GST activity, respectively, in erythrocyte suspension were found.
We found that >15 hours post-physical effort, CNVs' concentration was below the initial value, concomitant with other measured parameters: ChE and GST activity as well as IL-6 concentration, indicating a favorable effect of physical effort on health status. CNVs' concentration and ChE activity in isolates from peripheral blood proved to have potential as indicators of the response of the human body to inflammation after physical effort. Physical activity should be considered as an important factor in preparation of subjects for blood sampling in procedures focusing on CNV-containing diagnostic and therapeutic compounds.
Monolithic Convective Interaction Media (CIM) have been activated with epoxide and imidazole carbamate functionalities and used as supports for covalent immobilization of protein A, deoxyribonuclease ...I, and trypsin. The efficiency of immobilization for these proteins was determined from the amount of bound IgG, degradation of DNA, and hydrolysis of Nα‐benzoyl‐L‐arginine ethyl ester, respectively. The respective biological activities of trypsin and the binding capacity of protein A immobilized via imidazole carbamate groups were 11.45 and 2.25 times higher than those obtained for epoxide matrix while they were practically equal for deoxyribonuclease I. The kinetics of immobilization was studied in detail for trypsin under dynamic conditions and revealed that the enzyme immobilized via imidazole carbamate groups already reached its highest activity in 5 min. In contrast, a much longer time was required for immobilization via epoxy groups.
A deoxyribonuclease bioreactor was prepared by immobilization of deoxyribonuclease I through epoxy groups inherently present on poly (glycidyl methacrylate-co-ethylene dimethacrylate) monoliths. ...Columns with various levels of DNase activity were prepared varying immobilization temperature, pH, time and method. The apparent Michaelis-Menten constant, Km(app), and turnover number, k3app, for immobilized DNase determined by on-line frontal analysis method were, respectively, 0.28 g of DNA l(-1) and 16 dA260nm min(-1) mg(-1) of immobilized DNase. The highest activity of immobilized DNase was detected at 1 mM calcium ions concentration and mirrored properties of free enzyme; however, reaction temperature in the range from 25 to 37 degrees C has no significant effect on activity of immobilized DNase in contrary to free enzyme. The CIM DNase bioreactor was used for elimination of DNA contaminants in RNA samples prior to reverse transcription followed by PCR.
In
Aspergillus niger cells spontaneous posttranslational modification of 6-phosphofructo-1-kinase (PFK1) occurs. In a two step process the native enzyme (85
kDa) is first cleaved to an inactive ...fragment (49
kDa) that regains its activity after phosphorylation of the protein. The shorter PFK1 fragment exhibits changed kinetics, such as resistance to citrate inhibition. In order to avoid spontaneous complex posttranslational modification, modified gene was prepared encoding an active shorter PFK1 fragment. Since no appropriate microbial strains with disrupted native
pfkA genes were available,
Aspergillus niger strain with reduced likelihood for spontaneous posttranslational modification of PFK1 has been chosen for
in vivo tests. First, the appropriate length of a truncated gene was defined after a number of enzymes encoded by genes of different lengths had been tested. After adding sodium azide to the medium, phosphorylation was induced in the transformed hyphae to activate the shorter fragments which were subsequently screened for changed PFK1 kinetics. In the second step the responsible threonine residue was replaced with glutamic acid to elude the need for phosphorylation. An active shorter PFK1 fragment, resistant to citrate inhibition and activated to a higher level by fructose-2,6-bisphosphate with respect to the native enzyme was encoded directly from the modified gene.
The suitability of methacrylate based anion exchange monolithic supports for the separation and purification of plasmid and genomic DNA has been explored. The effect of the size of the channels, ...ionic strength of the solution, and ligand density on the dynamic binding capacity has been investigated. The dynamic binding capacity was found to be flow independent, at least up to a linear velocity of 700 cm h–1, and exceeded 9 mg mL–1 for all types of DNA. The recovery depends on the pH value of the mobile phase and its ionic strength as well as on the density of the active groups. Under optimal conditions recoveries exceeding 80% were obtained even for genomic DNA. Finally, the suitability of this approach is demonstrated by purification of a real‐life sample.
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