Overproduction of catalase by oxidative stress on Bacillus subtilis WSHDZ-01 / 生物工程学报

We studied the overproduction of catalase (CAT) by Bacillus sp.WSHDZ-01 by oxidative stress via the feeding of ethanol and the pulse addition of H2O2. By adding 2.0% (V/V) ethanol to the culture broth, the intracellular CAT activity reached 11 151 U/mL, which was 2.5 times than that of the control (4 450 U/mL in flask). By adding 0.3% (V/V) H2O2, more extracellular CAT secreted to the culture broth, and the ratio of extracellular CAT to the total CAT increased to 27%. Based on these results, an oxidative stress strategy combining the ethanol feeding and the pulse addition of H2O2 was developed. With this strategy, the ratio of extracellular CAT to the total CAT reached 82.5%, increased by 18.6% than that of the control (without ethanol and H2O2 addition). CAT production increased to 28 990 U/mL, which was 95.5% higher than the control (14 830 U/mL in 3 L fermentor). The fermentation time decreased to 42 h, which was much shorter than that of adding ethanol or H2O2, and CAT productivity reached 470 U/(mL x h) while the control achieved 396.4 U/(mL x h).

High-level production of alkaline polygalacturonate lyase in recombinant Pichia pastoris / 生物工程学报

In order to increase the production of alkaline polygalacturonate lyase (PGL) by recombinant Pichia pastoris GS115, the effect of cell and methanol concentration on the PGL production was carefully investigated by single factor experiment. The optimum conditions were listed as follows: the cell concentration 122 g/L, the methanol concentration 20 g/L, and the ratio of methanol and cell concentration 0.16-0.20 g/g (methanol/cell). With the glycerol and methanol feeding strategies, the ratio of methanol and cell concentration could be controlled at the range of 0.171 to 0.195 g/g. And the highest PGL activity (430 u/mL) and highest PGL productivity (4.34 u/mL/h) were achieved.

Response of bacillus sp. F26 to different reactive oxygen species stress characterized by antioxidative enzymes synthesis / 生物工程学报

The oxidative response of Bacillus sp F26 to different forms of reactive oxygen species (ROS) stress including H2O2, O2- * and OH * were investigated by using diverse generating source of ROS, which were characterized by synthesis of antioxidative enzymes. It was shown that the responses of cells to oxidative stress are largely dependent on species, mode (instantaneous and continual) and intensity of stress. Higher synthesis rate of catalase (CAT) is crucial for Bacillus sp F26 to resist H2O2 stress. The damage of H2O2 to cell was minor if CAT can efficiently decompose H2O2 entering into cell, furthermore, the response can stimulate cell growths and sugar consumption. Conversely, cell growth and synthesis of antioxidative enzymes are greatly inhibited when the intensity of H2O2 stress overwhelms the cell capability of clearing H2O2. Due to the difference in mode and effect on cells between O2- * and H2O2, higher synthesis rates of CAT and superoxide dismutase (SOD) couldn't guarantee cells to eliminate H2O2 and O2- * efficiently. Therefore, the toxicity to cells induced by intracellular O2- * is more severe than H2O2 stress. Unlike response to H2O2 and O2- *, OH stress significantly inhibited cell growth and synthesis of antioxidative enzymes due to the fact OH * is most active ROS. Our results indicated that Bacillus sp F26 will show diverse biological behaviour in response to H2O2, O2- * and OH * of stress due to the discrepancy in chemical property. In order to survive in oxidative stress, cells will timely adjust their metabolism to adapt to new environment including regulating synthesis level of antioxidative enzymes, changing rates of cells growth and substrate consumption.

Effects of Oxidative Challenge on Defensive Enzyme and Cofactors Level in Alkalibacterium sp.F26 / 微生物学通报

In this paper,a slight halophilic alkaliphile swain,Alkalibacterium sp.F26,which produced high level of intracellular CAT observed in previous research,was selected as a model microbial to explain the responses of this bacterium to oxidative stress.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 the aspects of defensive enzyme synthesis and cofactors level variation.As for catalase production,the activity increased up to 106.54 U/rag 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 H2P2 was 100 μmol/L.As far as energy state was concerned,ATP production and NAD+ generation were significantly inhibited from 20.55 μmol/L to 17.80 μmol/L and 69.89 μmol/L to 31.77 μmol/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 100 μmol/L H2O2,under which the activation of defensive mechanism resulted in an increase of ATE the level of ATP dropped from 22.69 μmol/L of the control to 22.38 μmol/L and 13.70 μmol/L when challenged by 50 μmol/L and 500 μmol/L H2O2.Besides,the concentration of NADH fluctuated and the NAD+ gradually reduced when H2O2 below 1 mmol/L.