Expression And Enzymatic Characteristics Of RNase R From Antarctic Psychrophilic Bacterium

Due to the special geographical location of Antarctica,the environment of cold,ultraviolet rays,oligotrophic,and high salt,provides conditions for the exploitation of cold-adapted enzymes resources in Antarctic microorganisms.These enzymes have high catalytic efficiency at low temperatures,so they have certain commercial value in the food industry and low-temperature biocatalysis.The research on the structure and functional characteristics of cold-adapted enzymes has attracted continuous attention in the field of molecular biology at home and abroad.Ribonuclease R(RNase R)participates in the RNA metabolism process of organisms and is widely used in scientific research and medical treatment.Moreover,its absence will cause the sensitivity of microorganisms to cold environments.Therefore,to enrich RNase R antarctic gene information and provide a scientific basis for further research,this study used antarctic sea-ice bacteria as the research materials,cloned and expressed the rnr genes to study the enzymatic properties of recombinant RNase R(r Ps RNR),as well as site-directed and deletion mutation of rnr gene.In this study,we used the Antarctic sea ice bacteria Psychrobacter sp.ANT206 as the experimental material,and cloned a new RNase R gene,named psrnr.Bioinformatics analysis of the psrnr gene using online websites found that it contained an open reading frame of 2313 bp and the molecular weight of Ps RNR protein was 87.42 k Da.After multiple sequence alignments,the catalytic sites of Ps RNR(Thr 646,Phe 654,Leu 665,and His 667)were found to be highly conserved.Using homology modeling to compare the structural models of Ps RNR and mesothermal homologues,it was found that electrostatic interactions(hydrogen bonds and salt bridges)and hydrophobic interactions were reduced,which would enhance the flexibility of Ps RNR and it was the reason for its high catalytic efficiency at low temperatures.p ET-28a(+)was used as an expression vector to connect psrnr gene amplicon.Subsequently,the recombinant vector was transduced into competent E.coli BL21 and the recombinant strain was selected.We designed primers for site-directed mutagenesis and used the Ni-NTA column to purify the r Ps RNR and mutant protein.The specific activity of r Ps RNR was 115.60?mol/min/mg,and the purification fold was about 5.58.The results of site-directed mutagenesis revealed that the active site His 667 played a crucial role in enzyme catalysis.Then,the enzyme properties of r Ps RNR were characterized by yeast RNA as a substrate.Studies on the enzymatic properties of r Ps RNR indicated that:r Ps RNR showed maximum activity at 30°C,retained 22.0-38.0%of its initial activity even at 0-10°C,and it explained thermal instability,these showed it was a cold-adapted enzyme;the optimal p H was 6.0 and r Ps RNR had excellent salt tolerance.In addition,r Ps RNR had a high k_cat value,and the?H value displayed a downward trend as the temperature increased.These characteristics will weaken the exponential temperature dependence of the reaction rate,which helped it to catalyze at low temperatures.Furthermore,conjugation of two steps was used to successfully construct the rnr gene deletion mutant strain?Ps RNR-ANT206.The genetic stability of the mutant strains and the survival rate under different temperature conditions were measured.We found that the deletion mutant exhibited good genetic stability.Additionally,the mutant strain was more sensitive to cold when the logarithmic strain was transferred to 4°C and 0°C,explaining that the rnr gene was very important for the growth of the strain at low temperatures.Overall,in this study,the molecular and enzymatic characterization of cold-adapted RNase R was carried out,and the importance of the rnr gene in the growth of microorganisms in cold environments was explored.It will lay the foundation for further research on the structure and functional properties of RNase R.