Studies On Gene Cloning, Expression And Optimization Of Fermentation Of Antarctic Microorganism SOD

Antarctica has its particular geogrpahical position and climate in which Antarctic microorganisms survived in it have formed very unique gene bank, genetic background and metabolic characteristics in the long process of evolution and growth. Studying on the mechanism of the Antarctic microorganisms adapted to extreme environment, excavating and developing its special functional genes is a hotspot of research in extreme microbes area. Superoxide dismutase(SOD) is a key enzyme for that it specifically catalyzes the harmful superoxide radicals(O2.-) to maintain the dynamic balance of the generation and elimination of reactive oxygen species(ROS) and reduce the risk of ROS damage and ensure that the organism could survive under the ROS pressure. Therefore, SOD plays an important role in biological adaption mechanisms to extreme environment.In this study, the strain Pseudoalteromonas sp. ANT506, which contained SOD was screened from the antartic microorganism by PCR amplification using a couple of degenerate primers. The target SOD gene, named Ps SOD, was connected with the p MD18-T carrier and then transformed into the cloning host E.coli DH5α. The Ps SOD was sequenced and its basic characteristic parameters were analyzed by a series of bioinformatics tools. The results showed that the Ps SOD had a complete open reading frame(ORF) of 582 bp with a putative product of 193 amino acid residues, an estimated molecular size of 21.4k Da, a p I of 5.09 and it was inferred as a hydrophilic protein. The analysis of structure of the protein showed that it was consisted of eight α-helix, three β-folding and random coils. The sequence analysis showed that the protein was belonged to Mn/Fe SOD with a conserved amino acid of WEHAYY. The sequence analysis showed that the protein was in related to Fe-SOD from Pseudoalteromonas haloplanktis TAC125 with a similarity of 97.9%. Further study showed that the protein was presumed a Fe-SOD with iron-cofactor binding sites: His18, His74, His167 and Asp162.The Ps SOD was connected with the p ET-28a(+) and then introduced into the E.coli BL21(DE3). The positive recombinant strain, named p ET-SOD, was selected and induced by IPTG at low-temperature(28oC) for heterologous expression. The expressed recombinationnal protein(r Ps SOD) was purified by Ni-chelating affinity and detected by SDS-PAGE. The results showed that the r Ps SOD was approximately 27 k Da. The analysis of of enzymatic characteristics showed that the special activity of the crude enzyme and the purified one was 46.6U/mg and 587.4U/mg, respectively. The r Ps SOD was puri?ed 12.6-fold by Ni-af?nity chromatography with a yield of 22.9%. The puri?ed r Ps SOD was found a maximum activity at 30 oC, p H8.0 and exhibited 13.9% activity at 0oC. Compared with some other SODs about enzymatic actibities, catalytic activity at low temperature and thermal instability of r Ps SOD incidated it was a coldadapted enzyme. r Ps SOD exhibited well capability to 2.5M Na Cl(62.4%) and other inhibitions, which meaned that r Ps SOD had certain ability of salt stress tolerance. The r Ps SOD had its unique enzymatic characteristics, which may be associated with the environment of the Antarctic sea ice at low temperature and high salt.The fermentation medium of r Ps SOD was studied by Design-expert. The result showed that lactose, tryptone and tween-80 were the most important factors to affect microbial growth and the activity of r Ps SOD. A quadratic regression model of r Ps SOD activity was built and the optimal fermentation conditions for Tween-80 0.047%, tryptone 6.16 g/L, lactose 11.38 g/L, yeast 5 g/L and Na Cl 10 g/L with a p H of 7.5 were verified and determined, which would provide vital parameters for SOD’s large-scale fermentation.In this research, SOD from Antarctic cold-adapted microorganisms was systematically researched by gene cloning, expressing, enzymology characteristics and recombinant bacteria fermentation. This study was aimed at gaining novel-functional genes with independent intellectual property rights and providing theoretical basis for development and application of cold-adapted enzyme from Antarctic microorganisms.