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    Cloning and expression of the Vitreoscilla hemoglobin gene in Enterobacter aerogenes
    (Pleiades Publishing Inc, 2004) Erenler, SO; Gencer, S; Geckil, H; Stark, BC; Webster, DA
    The hemoglobins found in unicellular organisms show a great deal of chemical reactivity, protecting cells against oxidative stress, and hence have been implicated in a wider variety of potential functions than those traditionally associated with animal and plant hemoglobins. There are well-documented studies showing that bacteria expressing Vitreoscilla hemoglobin (VHb), the first prokaryotic hemoglobin characterized, have better growth and oxygen uptake rates than their VHb counterparts. Here, the expression of VHb, its effect on the growth and antioxidant enzyme status of cells under different culture conditions was studied by cloning the complete regulatory and coding sequences (vgb) for VHb in Enterobacter aerogenes. Contrary to what has been reported for Escherichia coli, the expression of vgb in E.aerogenes decreased several fold under 10% of atmospheric oxygen (approximate to2% oxygen) and its growth was not greatly improved by the presence of VHb. Measured either as viable cells or total cell mass, untransformed E. aerogenes grew better than the recombinant strains. At the late exponential phase, however, the vgb-bearing strain was determined to have a higher cell number and total cell mass than the strain bearing only the plasmid vector with no vgb insert. The VHb expressing strain also had an oxygen uptake rate several fold higher than its counterparts. Given that oxidative stress may occur upon elevated oxygen exposure and be balanced by the action of antioxidative compounds, the level of antioxidative response of E. aerogenes expressing VHb was also studied. The VHb expressing strain had substantially (1.5-2.6-fold) higher catalase activity than strains not expressing VHb. Both VHb+ and VHb- strains, however, showed similar levels of superoxide dismutase activity. The activity of both enzymes was also growth phase dependent. Stationary phase cells of all strains showed 2-5-fold higher activity for these enzymes than cells at the exponential phase.
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    Genetic engineering of Enterobacter aerogenes with the Vitreoscilla hemoglobin gene
    (Elsevier, 2003) Geckil, H; Gencer, S; Kahraman, H; Erenler, SO
    Hemoglobins in unicellular organisms, like the one here in the bacterium Vitreoscilla, have greater chemical reactivity than their homologues in multicellular organisms. They can catalyze redox reactions and may protect cells against oxidative stress. The ability of Vitreoscilla hemoglobin to complement deficiencies of terminal cytochrome oxidases in Escherichia coli also suggests that this hemoglobin can receive electrons during respiration. In this study, a recombinant strain of Enterobacter aerogenes engineered to produce the Vitreoscilla Hb was investigated with regard to its susceptibility to oxidative stress. The culture response to oxidative stress produced by exogenously applied hydrogen peroxide was characterized in terms of cell growth, survival and the activities of two key antioxidant enzymes (catalase and superoxide dismutase). The influence of the physiological state of the cells and different media upon these culture dynamics was determined. Results showed that the hemoglobin-expressing strain is quite distinct in terms of growth/survival properties and activity of antioxidant enzymes from that of non-hemoglobin counterparts. (C) 2003 Editions scientitiques et medicales Elsevier SAS. All rights reserved.
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    Membrane permeabilization of gram-negative bacteria with a potassium phosphate/hexane aqueous phase system for the release of L-asparaginase: an enzyme used in cancer therapy
    (Elsevier Sci Ltd, 2005) Geckil, H; Ates, B; Gencer, S; Uckun, M; Yilmaz, I
    A fast, efficient and reproducible recovery procedure for periplasmic L-asparaginase from two distinctly related gram-negative bacteria, Enterobacter aerogenes and Pseudomonas aeruginosa, is presented. As the method uses inexpensive organic solvent hexane and an aqueous salt solution, it is also highly cost-effective in comparison with the currently available techniques used for the release of this enzyme. As hexane is a highly water immiscible organic solvent, it can be removed easily from the top of the aqueous phase by a simple evaporation. Also, various organic solvents and other membrane partitioning compounds were compared for their efficiency on L-asparaginase/protein release. The degree to which the enzyme was released was different for two bacteria, suggesting that they possess different permeability characteristics. The most efficient enzyme release from both bacteria was determined to be in 50 mM potassium phosphate with 1% hexane. Enzyme recoveries up to three-fold with respect to sonication have been achieved with this system. (C) 2004 Elsevier Ltd. All rights reserved.
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    Production of L-asparaginase in Enterobacter aerogenes expressing Vitreoscilla hemoglobin for efficient oxygen uptake
    (Springer-Verlag, 2004) Geckil, H; Gencer, S
    This study is the first utilizing Vitreoscilla hemoglobin in a heterologous bacterium, Enterobacter aerogenes, to determine the effect of such a highly efficient oxygen-uptake system on the production of L-asparaginase, an enzyme that has attracted considerable attention due to its anti-tumor activity. Here, we show that the Vitreoscilla hemoglobin expressing strain has from 10-fold to more than two orders of magnitude lower L-asparaginase activity than the wild type or the control without the Vitreoscilla hemoglobin gene under different aeration conditions. Aeration and agitation were also determining factors for enzyme production. The enzyme activity was reduced considerably under both full aerobic and anaerobic conditions, while the highest enzyme activity was determined in cultures under low aeration and low agitation. Also, the effect of different concentrations of glucose on enzyme production showed catabolic repression. Glucose at 1% caused almost total inhibition of enzyme activity, while at 0.1% it showed a slightly stimulatory effect on enzyme production, compared with glucose-free medium.
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    Vitreoscilla hemoglobin expressing Enterobacter aerogenes and Pseudomonas aeruginosa respond differently to carbon catabolite and oxygen repression for production of L-asparaginase, an enzyme used in cancer therapy
    (Elsevier Science Inc, 2004) Geckil, H; Gencer, S; Uckun, M
    The production of antileukemic enzyme L-asparaginase in two distinctly related bacteria, Enterobacter aerogenes, Pseudomonas aeruginosa, and in their recombinants expressing the Vitresocilla hemoglobin (VHb) has been studied. Both bacteria showed a substantially different degree of carbon catabolite repression of the enzyme production. E. aerogenes grown under catabolite repression had more than 20-fold lower L-asparaginase activity than the controls. This figure was only 1.6-fold for P. aeruginosa. In the medium with restricted nutrient content, however, the inhibitory effect of glucose on the enzyme production was less pronounced. The presence of VHb, an efficient oxygen uptake system, had also different effects in both bacteria. Under conditions of no catabolite repression, this protein caused about 7-fold lower L-asparaginase activity in E. aerogenes, but similar or even slightly stimulatory effect in P aeruginosa. The use of a relatively poor carbon source, mannitol, caused a lower L-asparaginase level and no glucose type catabolite repression. (C) 2004 Elsevier Inc. All rights reserved.
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    Vitreoscilla hemoglobin renders Enterobacter aerogenes highly susceptible to heavy metals
    (Springer, 2004) Geckil, H; Arman, A; Gencer, S; Ates, B; Yilmaz, HR
    When expressed in heterologous microorganisms Vitreoscilla hemoglobin (VHb) acts as oxygen storage and causes a higher oxygen uptake. In this study, the effect of this protein on growth, sensitivity and antioxidant properties of Enterobacter aerogenes exposed to metal stress was investigated. The strain expressing VHb was more sensitive to mercury and cadmium as the minimal inhibitory concentration (MIC) for these metals was up to 2-fold lower in this strain than the host and the recombinant strain carrying a comparable plasmid. At lower concentrations than MIC, the metals partially limited growth and caused an inhibition proportional to metal concentration applied. The growth pattern of VHb expressing strain was also distinctly different from other two non-hemoglobin strains. The hemoglobin containing strain showed substantially higher superoxide dismuates (SOD) activity than the non-hemoglobin strains, while catalase levels were similar in all strains. All strains exposed to copper, however, showed similar MIC values, growth patterns, and SOD and catalase levels.