Study On Glutathione And Glutathione-Related Enzymes Of Antarctic ICE Microalgae GJ01 And ICE-L

The Antarctic microorganism can survive in extreme environment after long period of adaptation to extreme environment, and has given rise to people' s greatly attention since 1970' s, and becomes one of hot research field at present. Antarctic ice alga is one of main producers of primary production in marine ecology system of Antarctic pole region, and takes up important status and plays important role in Antarctic ecology system. GSH and GSH-related enzymes play a great role in protecting organisms from oxidative damage caused by ROS, including Glutathione (GSH), Glutathione peroxidases (GPx), glutathione S-transferases(GST), and Glutathione reductase (GR). Ice microalga living in extreme environment ,which characterized by low temperature , high dissolved oxygen, presence of ice and strong seasonal changes in light intensity, are investigated more and more in recent years. They should have a more effective mechanism to remove ROS.In this study, GSH level in the ice alga cells and effects on the GSH level were assessed, as well as adaptation mechnism of GSH and GSH-related enzymes to low temperature and high metal was investigated. GSH product of ice algae increased through the optimization of the culture, and Antarctic ice alga would be a new resource of GSH. GR of ice algae was purified , and its characters were elucidated as well. These results make the adaptation mechanism of ice alga clarified more all-around than before, which can provide new idea for the improvement of anti-disadvantageous environment organism species through gene engineering method. All theresearch were summarized as follows.(1) GSH content, GSH produce ability and GR activity in more than 20 Antarctic ice microalgae , isolated in five different phylum from Antarctic seawater and Antarctic ice samples, were assessed using the spectrophotometer methods. The results showed that GSH content of Cyanophyceae B-l is highest, and GPA of Antarctic ice microalgae was higher than the control. In most Antarctic ice microalgae GR activity was greater than the control microalgae , including B. rut Hans GJ01 and Chlmydomonas sp. ICE-L. B.rutilans GJ01 grew more quickly in f/2 medium, but Chlamydomonas sp. ICE-L in Provasoli medium. These make it possible that Antarctic ice microalgae will become a new resource of GSH.(2) The growth curve, change of GSH with growing and effect of adding medium method on GSH content were studied in B. rut Hans GJ01, as well as GPA and GR during the growing. It was showed that growth cycle of B. rutilansGJOl is about more than 14 days, and the logarithm phase of growth is between day 6 and day 10. GSH content, GPA and GR activities changed periodically during the growth. GSH content is highest in logarithm anaphase and early time of stable phase, which is suit to gain GSH. When adding the new medium to the cultures , which has grown from 6 to 8 days, GSH content, GPA activity and protein concentration of ice microalgae would increase. These will provide a guide on the application of Antarctic ice microalgae practically.(3) Effects of nutrition factors and amino acid contributed to GSH on the growth and GSH product of B. rutilans GJ01 were studied, as well as the optimization of synthesis condition through the whole cells of B. rutilans GJ01 and free-cell enzymes combined with ATP regenerative system of B. rutilans GJ01. Vitamin B12 and biotin had no large effect on GSH product. Optimum temperature of GPA is 10 —25°C, and optimum pH of that is 7.0. Optimum concentration of amino acid Glu, Cys, Gly is 24mmol/L> 12mmol/L^ 8 mmol/L respectively with only factor test.GPA of B. rutilans GJO1 is dependent on Mg2+, optimum concentration of which is 10 — 20 mmol/L. It is disclosed through the orthogonal test that the most optimum condition for GSH product of whole ice algae B. rutilans GJ01 is 16h light/8h dark, 1. 6g/L NaHC03, 0. 4 mmol/L Ca2+and 10 mmol/L Cys. Like above, the most optimum condition of GSH product is 30°C, lOmmol/L Cys, 30mmol/L Mg2+, pH 7.5 using method of the free-cell enzymes combined with ATP regenerative system of B. rutilans GJ01.(4) The possible GSH system of Chlamydomonas sp. ICE-L of adaptation to low temperature has been investigated by evaluating GSH and GSH-related enzymatic responses at different temperatures using the spectrophoto-meter methods, as well as the re-acclimation to optimal temperature of Chlamydomonas sp. ICE-L , which have acclimatized the temperature which was higher than the optimal temperature. The results showed that GSH content, GPx> GST and GR activities were higher at 0°C and —10°C than 8°C, but the protein concentration decreased. The same results appeared in re-acclimation test. GSH content, GST and GPx activities droped when Chlamydomonas sp. ICE-L were taken from 8°C to either 12°C or 17°C , but protein concentration and GR activity would elevate. It is suggested that the GSH defence system is correlated positively with the low temperature and other factors but GR are correlated negatively with high temperature. In a word, GSH and GSH-related enzymes play an important role in the adaptation of Antarctic ice microalgae to the low temperature.(5) The GSH content and GSH-related enzymes were investigated as well as the growth yield and malonyldialdehyde (MDA) content in Chlamydomonas sp. ICE-L exposure to the different cadmium concentration, which is one of the most toxic and general metal pollution and could appear in the Antarctic pole region with the development of human activity. The results showed that the higher concentration Cd inhibited the growth ofChlamydomonas sp. ICE~L significantly and Cd would induce formation of MDA. At the same time, it is clear that GSH content, GPx and GST activities were higher in Chlamydomonas sp. ICE-L exposed to Cd than the control. But GR activity dropped notably when Chlamydomonas sp. ICE-L were cultured in the medium containing Cd. Increase of GSH content and GPx and GST activities acclimate to oxidative stress induced by Cd and protect Antarctic ice microalga Chlamydomonas sp. ICE-L from toxicity caused by Cd exposure. The interaction of metals with GSH is an integral part of the toxic response. It is possible that Cd can inhibit the activity of GR in vivo. It is observed clearly through ultrastructure that starch granule and chloroplast were destroyed in a little grade, and vacuole increased and contracted, and protoplasm shrinked. Distortion of membrane and slur cell wall with crack were found at the same time,which had negative effect on the growth and metabolism of ice microalgae. These parameters may be used to assess the biological impact of Cd on the Antarctic pole region environment.(6) GR of Chlamydomonas sp. ICE-L was purified by 12 771 fold using precipitate of saturation(NH4)2S04, iron exchange , affinity chromatography and gel filtration. It is showed that the product is homogeneity in terms of SDS-PAGE electrophoresis, and its specific activity is 178.8000 U/mg protein, and its yield reachs 25.1%. The apparent molecular weight is 54. 6kDa like other GRs. Apparent KmuADPHof GR is 23. 3n mol/L, which is higher than the others, and apparent KntesG is 66. 0 u mol/L, but apparent Km^ is 83. 8 li mol/L. It can depend on NADH as electronic donor.(7) Properties of Chlamydomonas sp. ICE-L GR were investigated thoroughly depending on the purification above using the method of enzyme activity assessment. Optimum pH of it is 7. 5, and it is stable to pH except for the high basicity. But it is unstable to higher temperature, and its optimum temperature is 25 °C . Its apparent activity energy is3. 7kJ/(mol ? K), and it is suggested to conserved at —78°C. Its optimum buffer, iron strength and Mg2+ is 50mmol/L Tris~HCl (pH7. 5), 90 mmol/L and 7. 5mmol/L respectively. (NH4)2SO4(1 mmol/L), Ca2+, Mg2+, GSH, Cys and ATP can induce activity of GR, but the chelatin , high metal such as Cd2+ , Pb2+ , Cu2+, Zn2+ and so on, surface active agent, enzyme protective additive, NEM, NADPH and ADP inhibited the activity of the GR.It is a low temperature enzyme, and is important for the study on adaptation and catalyzing mechanism of low temperature enzymes .In a word , the acclimation mechanism to disadvantageous conditions of Antarctic ice microalga has formed after very long period of evolution. GSH system plays a great role on protecting ice algae from low temperature and high metal. Increase of GSH and GSH-related enzymes is helpful to scavenge ROS and detoxification, which is a part of all adaptation mechanisms. Optimization of GSH synthesis condition of ice microalga , which contain higher GSH content, makes it possible that people produce GSH using them in practice. GR from Antarctic ice microalga Chlamydomonas sp. ICE-L is a low temperature enzyme, and has sameness and variance compared to other GRs. The study of this special GR has great signality on the resource of GR and adaptation mechanism of other low temperature enzymes like GR to low temperature.