Studies On Gene Expression And Enzymatic Properties Of Antarctic Sea Ice Bacteria SHMT

Antarctica is a place with low temperature,high salt and strong radiation all the year-round.In such a harsh environment,the microorganisms that survive have evolved over a long period of time to adapt to the low-temperature environment,thus forming cold-adapted microorganisms.Serine is an essential amino acid that catalyses the conversion between serine and glycine by serine hydroxymethyltransferase(SHMT)(E.C.2.1.2.1).In this paper,we firstly screened a shmt gene containing strain Psychrobacter sp.ANT206(P.sp.ANT206)from Psychrobacter sp.Primers were designed based on the genomic DNA of P.s.ANT206,and the genomic sequence of the target gene(named as psshmt)was obtained by PCR amplification.T-A cloning of psshmt was performed to screen the positive cloned strains,and then the sequence was determined.The protein sequences of Ps SHMT and Psychrobacter aquaticus were the most similar at 95.93%.Analysis by NCBI,Bio Edit,et al,showed that Ps SHMT protein had 4 active sites,13glycine-pyridoxal phosphoric acid binding sites,and 7 folic acid binding sites.The psshmt gene was linked to pet-28a(+)and transferred to the host bacterium E.coli BL21(DE3)for expression.Positive recombinant bacteria(named pET-shmt)were screened,and a large amount of recombinant bacteria were induced and expressed.Recombinant protein(rPsSHMT)was purified by Ni-NTA and identified by SDS-PAGE.The results showed that rPsSHMT was about 45 k Da.Enzymatic analysis showed that the optimum temperature of rPsSHMT was 30°C.The enzyme was most active at p H 7.5;Ca²⁺could increase the activity of rPsSHMT.The maximum value of k_catat rPsSHMT was 20.28 1/s at 30°C,and rPsSHMT had lower?G than the other mesophilic enzyme,which further indicated rPsSHMT was a cold-adapted enzyme.The functional groups of rPsSHMT were studied and arginine residues were found to be the active central group of the enzyme.Compared with homologous mesophilic enzymes,it can be seen that the cold-adapted rPsSHMT had a relatively low content of proline residues and a small number of hydrogen bonds in terms of structure.These structural characteristics gave the protein molecules high flexibility,making it more active at low temperatures.The fermentation conditions of rPsSHMT were optimized by response surface method,and the optimal fermentation conditions were determined as follows:induction temperature was 14°C,induction time was 8 h,12 m M DL-3 phenylserine,0.05 m M PLP,0.05 m M Na Cl,1 m M IPTG,1 g/L Na₂SO₄,7.5 g/L tryptone,and 3 g/L yeast extract.Under this condition,the optimal rPsSHMT specific activity was 67.00 U/mg,and the specific activity of rPsSHMT was increased by 1.51 times after optimization compared with before optimization,indicating that the fermentation optimization model established in this study provided the reference for large-scale fermentation of recombinant protein and has a certain value.This study provided a theoretical basis for the development and application of cold-adapted enzyme from Antarctic microorganisms.