Expression And Enzymatic Characteristics Of GST And SOD From Antarctic Psychrophilic Bacterium

Antarctic sea ice is characterized by low temperature,strong ultraviolet radiation and high salinity because of its unique geographical location.These characteristics can cause superoxide anion radicals(O^2-.)and reactive oxygen species?ROS?to accumulate in organisms and lead to oxidative stress.Therefore,in order to adapt to the Antarctic sea ice environment,Antarctic organisms can eliminate oxidative stress caused by the external environment protects cells by producing low-temperature antioxidant enzymes.Glutathione S-transferase?GST?and superoxide dismutase?SOD?are two important antioxidant enzymes in organisms,which have broad application prospects in cosmetics,medicine and low-temperature food antioxidants.Therefore,this paper used Halomonas sp.ANT108 to study its gst and sod genes in order to provide a theoretical basis for the future development of low temperature antioxidant enzymes from Antarctica.In this paper,the gst gene of Antarctic sea ice bacteria Halomonas sp.ANT108 was cloned and analyzed.The results showed that the gene was 603 bp,named hsgst,and can encode a protein containing 200 amino acids,named Hs GST.The predicted molecular weight was 21.76 k Da and the theoretical isoelectric point?p I?was 5.76.Multiple sequence alignment showed that there was an H site and an N-terminal domain in the sequence of Hs GST.The results of homology modeling showed that compared with Ec GST,Hs GST exhibited many characteristics of cold-adapted enzymes,which increased the flexibility of protein structure and ensured high catalytic efficiency at low temperature.The enzymatic properties of the purified r Hs GST were studied.The optimum temperature of r Hs GST was 25 ?,and the activity of r Hs GST was 41.67 % at 0 ?.However,r Hs GST had thermolability,and it became denatured and inactivated after 80 min incubation at 40 ?.The optimum p H value of r Hs GST was 7.5.In addition,r Hs GST had a higher substrate affinity for GSH than for CDNB.The results of paper diffusion method showed that r Hs GST could protect cells from oxidative stress induced by hydrogen peroxide and had antioxidant effect.The sod gene in Halomonas sp.ANT108 was also studied.The sod gene was 708 bp,named hssod,and its encoded protein was named Hs SOD,which contained 235 amino acids.The predicted molecular weight was 25.04 k Da,and the p I was 5.17.The results of multiple sequence alignment showed that there was an active site,Cu²⁺ binding site,Zn²⁺ binding site and a dimer interface in the sequence of Hs SOD.According to the results of phylogenetic tree and type identification experiments,it was found that the auxiliary metal ions bound by hssod were Cu²⁺ and Zn²⁺,belonged to Cu/Zn SOD.Compared with mesophilic Bs SOD,Hs SOD also exhibited the characteristics of cold-adapted enzymes,such as higher frequency of glycine residues,less interaction between hydrogen bonds and salt bridges.It was found that the optimum temperature and p H of r Hs SOD were 35 ? and 8.0,respectively.And r Hs SOD had thermolability.Thus,it was a cold-adapted enzyme.When the salt concentration was 1.5 M,the activity of r Hs SOD could maintain 50 % of the initial enzyme activity,which indicated that r Hs SOD had a certain ability to resist salt stress.The above systematic study of two kinds of antioxidant enzymes from Antarctic sea ice bacteria.It will be helpful for further understanding of Antarctic cold-adapted enzymes,and has important scientific research significance and potential application value for the development of independent intellectual property rights extreme functional gene resources.