Serralysin Of Serratia Sp. FS14 IS A Bifunctional Protein With Protease And Dnase Activity

Previously, we isolated a serratia strain Serratia.sp FS14 from the stem of the diseased Atractylodes macrocephala Koidz. Our previous result showed that the 50kDserralysin secreted by Serratia sp. FS14 is a thermostable bifunctional protein with both protease and DNase activity.To further characterize the 50kD serralysin protein from FS14, we purified the 50kD protein from the FS14 strain and characterized it. Afterwards, we amplified the gene encoding for 50kD serralysin by PCR using FS14 genomic DNA as template, and cloned it into the expression vector pET24b. Heterologous expression was carried out in E.coli. The expression products were purified after denaturation and renaturation process. The catalytic activity analysis of the refolded protein showed that it had both protease activity and DNase activity. Moreover, we crystallized the 50kD serralysin protein and solved its structure. By comparison with the structure of serralysin reported, we found that our structure is almost identical with the previously reported structure and only shows little difference in a loop region.By homologus recombination method, we know out nucA and 50kD serralysin gene respectively. Then we analyzed the DNase activity of the secreted protein of the mutants. The result showed that the supernatant of the nucA mutant do still contains thermostable DNAse activity, while the supernant of the 50kD serralysin deletion mutant contain both nuclease activity and thermostable Dnase activity. By comparison of the protein pattern of the supemant of the wild and the mutant, we found that there is another 50kD protein left in the supernatant of 50kD serralysin mutant, so we further purified this protein from the 50kD serralysin gene deletion mutant strain. The enzyme activity assays showed that it also had both protease activity and DNase activity. Mass spectrometry result showed that it also belonged to serralysin.Analysis of the genome sequence of FS14, we found four different genes encoding for serralysin like protein and named them as 50kD serralysin, serralysin-protease like A、B、C. To characterize serralysin-protease like A、B、C, we amplified the genes encoding for serralysin-protease like A、B、C by PCR using FS14 genomic DNA as template, and cloned into the expression vector pET24b. Heterologous expression was carried out in E.coli, and the expression products were purified after denaturation and renaturation process as 50kD serralysin. The catalytic activity analysis of the refolded proteins showed that these three proteins also have both protease activity and DNase activity. So, we conclude that the serralysin family proteins from FS14 are bifunctional proteins with both protease activity and DNase activity.In order to identify the DNase active site of the 50kD serralysin protein of FS14, we constructed a series of single mutants by site-directed mutantion. The DNase activity assay of these mutants showed that the mutation of some sites could decrease the DNase activity of this protein significantly. Besides, most of these sites are located between residue 185 and 195 which forms a loop domain, this loop domain is different between our structure and the previously reported structure.