| Reactive oxygen species (ROS), including superoxide radicals(O2 - ), hydrogen peroxide(H2O2)and hydroxyl radicals(·OH)are a group of activated molecules that continuously formed during normal aerobic metabolism in micro-organism. The condition known as oxidative stress results when the production of ROS exceeds the capacity of the cells to remove them safely. In the condition of oxidative stress, ROS can oxidize protein, nucleic acids and lipid peroxidation, lead further to disruption of normal metabolism, mutations, and ultimately cell death. To protect cells against ROS, all aerobic micro-organism instinctively respond to the stress using enzymatic and non-enzymatic defence systems, and thus show different biological phenomenon. Study the mechanism of cells response to oxidative stress is helpful to understand the causation of human disease caused by ROS and find effective means of protection and clearance.In this paper, the physiological mechanisms to oxidative stress of a slight halophilic alkaliphile strain, Alkalibacterium sp.F26, which produced high level of intracellular CAT were investigated. The main contents of this dissertation included as follows:(1) The effect of H2O2 instantaneous stress on CAT synthesis through single addition of H2O2 in shake flasks was studied. It was found that influence of H2O2 stress on cell growth and CAT synthesis was largely dependent on cell growth phase. Initial addition of H2O2 was not advantageous for CAT production, and no obvious bad effects of cell growth were observed. Significant enhancement of CAT activity was observed by H2O2 stimulation when cell growth enter exponential phase (18 h), although cell growth was inhibited by H2O2 addition. The activity of CAT increased to 94.56 u/mg protein with addition of 3 mM H2O2. However, the normal metabolism was badly damaged, casusing CAT level rapidly decreased when the concentration of H2O2 is 4 mM.(2) The effect of H2O2 on cofactors level was also carefully investigated by using HPLC. The results indicated that Alkalibacterium sp. F26 had obvious responses to higher concentration (>1 mmol/L) of H2O2 than that to lower H2O2 (<1 mmol/L) challenge from those aspects of defensive enzyme synthesis and cofactors level variation. As for catalase production, the activity increased up to 106.54 u/mg protein, which was 1.76 fold of the control when cells were challenged by 3 mmol/L H2O2, but its activity only was 1.13 fold when H2O2 was 100μmol/L. As far as energy state was concerned, ATP production and NAD+ generation were significantly inhibited from 20.55μmol/L to 17.80μmol/L and 69.89μmol/L to 31.77μmol/L, respectively, leading to the drop of energy charge from 0.77 to 0.68 and the increase of the portion of NADH/NAD+ from 0.08 to 0.41 in the former case. However, these effects were less distinct under lower concentration of H2O2. Except of the condition of 100μmol/L H2O2, under which the activation of defensive mechanism resulted in an increase of ATP, the level of ATP dropped from 22.69μmol/L of the control to 22.38μmol/L and 13.70μmol/L when challenged by 50μmol/L and 500μmol/L H2O2. Besides, the concen-tration of NADH fluctuated and the NAD+ gradually reduced when H2O2 below 1 mmol/L.(3) The influences of H2O2 on key enzymes of glucose metabolism were studied. The res- ults shown that G6PD of PPP was induced with the increasing extent of oxidative stress. In the case of HK and PK of EMP, both of their activity dropped because of oxidation of sulfhydryl-group. Besides, SDH of TCA level increased under trace concentration of H2O2 to maintain normal metabolism of cells. However, inactivation of SDH occurred due to oxidation of active site, iron-sulfur, when exposed to high stress.
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