Purification And Characterazation Of A Secreted Dnase From Exiguobacterium Sp.yc3

A nuclease is a phosphodiesterase that cleaves phosphodiester bonds in the nucleic acid.Nucleases widely exist in plants,animals and microbes and play important roles in DNA replicating,proofreading,recombinating and some other important activities.In this study,a bacterium strain with extracellular thermostable DNase was isolated from the Changjiang River at Yichang and identified as Exiguobacterium sp.yc3.To further study the thermostable DNase,a 20 kDa protein with DNase activity was isolated from the culture supernatant by ammonium sulfate precipitation,anion chromatography and cation chromatography sequencially.The optimum pH for the DNase activity is from 6 to 9 and the optimum temperature is from 28?-37?.The result also showed that this DNase is thermostable after pretreatment at 90 ? for 10 mins.Mass spectrum was used to determine the protein sequence,and the Mascot peptide fragments search result showed that matching peptide fragments accounted for 17%(37/210)of the amino acid sequences of a secreted protein from E.sibiricum 255-15.The secreted protein from E.sibiricum 255-15 was then used to search for its homologous proteins and the degenerate primers for amplifying the putative DNase was then designed according to the result of nucleotide sequences alignment based on the searched homologous proteins.The gene of the putative DNase was amplified and sequenced,the protein BLAST showed that the putative DNase belonged to HNH nuclease superfamily.This putative DNase was then designated EheA(Exiguobacterium HNH Endonuclease).The recombinant EheA was successfully expressed by fusion to the maltose binding protein.The mature recombinant EheA was then purified to homogenous after removing the MBP tag by using TEV protease The DNase activity analysis showed that the mature recombinant EheA exhibits similar properties as the native EheA isolated from the cultural supernatant.In conclusion,EheA is a thermostable DNase.To clarify the active site of the EheA,protein sequence alignment of EheA with the 500 top score homo logs was performed.The result showed that among these residues,the H116,N141 and N156 are completely conserved.To check whether these conserved residues constitute the HNN motif,site-directed mutants of these residues were constructed respectively.The results showed that all mutant proteins lost their DNase activity,which indicates that the H116,N141 and N156 are essential for the DNase activity and suggests that EheA may be classified into the second subfamily of the HNH superfamily which has a typical conserved HNN motif in the sequence.We also confirmed that H64 in EheA is needed for the DNase activity by the site-directed mutagenesis.In addition,we find that EheA homologs are universally conserved in the genome of Exiguobacterium species and the isolates containing EheA genes have DNase activities.Moreover,as the EheA genes are conserved and the phylogenetic analysis result of EheA homologous is coincided with the classification of the Exiguobacterium species,EheA may play important roles in the Exiguobacterium species.