Isolation, Purification And Characterization Of Two Secreted Proteins In Serratia Sp.FS14

Serratia.sp FS14was previously isolated in our lab from the stem of the diseased Atractylodes macrocephala Koidz. Themostable extracellular DNase and protease were detected in the supernatant of the culture of Serratia.sp FS14while the FS14was a mesophile. This is the first report of themostable extracellular enzymes in Serratia.In order to isolate and characterize this themostable DNase, two secreted proteins, about50Ku and40Ku, were purified from the supernatant of the culture by ammonium sulfate precipitation, DEAE cellulose chromatography and Superdex G-200gel filtration. The50Ku protein was then detected as a DNase after the purification. But we were surprised to find that the50Ku protein was a previously reported Metalloproteinase after amino acid sequence determination by the Mass Spectrometry. The following protease activity analysis showed that it does have thermostable protease activity. To verify this50Ku protein was a bifuctional protein, the gene encoding for the50Ku protein was amplified from the genomic DNA of the Serratia.sp FS14according to the reported conserved sequences of the Metalloproteinase. Then the gene was cloned into expression vector pET24b and the recombinant plasmid pET24b-50Ku was then expressed in the host strain E. coli C43(DE3). A50Ku extra protein band could be detected on the SDS-PAGE gel, but this protein was detected in the form of inclusion body. So denaturation and renaturation process of the inclusion body was performed to get the correctly folded protein. The refolded protein was verified to have both DNase and protease activity. In addition, a mutant with the catalytic residue E174A of the protease was obtained by site mutagenesis. The catalytic activity analysis showed that mutant loss protease activity but still has DNase activity. Acoording to the above result, we conclude that this50Ku protein is a bifunctional protein with DNase and protease activity.The40Ku protein was identified as Flagellin after amino acid sequence determination by the Mass Spectrometry, The gene of Flagellin was then amplified from total DNA of Serratia.sp FS14by PCR according to the reported conserved sequences. The gene was sequenced and the homologous analysis showed that there was83%homology with the reported amino acids of Flagellin of Serratia marcescens(ACCESSION:P13713).