Isolation, Functional Characterization And Gene Cloning Of A Metalloprotease Production By Serratia Nematodiphia

Metalloprotease is one of the kinds of protease which activity center depends on metal ion.Metalloptease is a ubiquitous protease in nature which has some different characters, so they have significant value in economy and applications. Currently, metalloprotease have been applied in foodstuff, scour, cosmetic, pesticide and anti-tumour druggery. However, the resources of the metalloproteinase had been found are not rich enough which limited its further utilization. So it is significant to find more microbial metalloproteinase.A pathogen strain Serratia nematodiphila was isolated from intestine of Entomopathogenic nematodes (EPNs) and has been identified. The results of testing the proteolytic activity of Serratia nematodiphila culture filrate showed that the strain Serratia nematodiphila has remarkable extracellular proteolytic activity. The protease from Serratia nematodiphila was purified by Sepharose G-75 gel filtration chromatography, DEA-Sepharcyl Fast Flow ion-exchange chromatography, resource Q FPLC and denaturalization SDS-PAGE method. The final yield was about approximately 8 mg protease in 1L ferment filtration. The purified protease migrated as a single band on SDS-PAGE with estimated molecular weight of 50 kDa.Extracellular proteolytic activity was preliminarily assayed by the casein agar plates on which S. nematophila has been inoculated showed evident clearance halos indicative of protein degradation. And the change of the level of proteolytic activity in response to the growth of S.nematophila was clarified by measured the value of the clearance halos.The extracellular protease activity increased with the growth of the strain in LB culture medium and the protease activity reached maximum at the exponential growth phase later. The variation of the extracellular protease activity when the strain grown in minimal culture medium added with casein protein was similar to it grown in (?) cuiture. The only difference is that the time of the extracellular protease activity reached maximum was advanced about 8 h compared with in LB culture medium. Through compared the growth and the protease activity of culture the strain in different conditions, we determined the style of the strain secreted extracellular protease shoud be inducible expression.The first 15 amino acid residues of the protein were determined and the sequence was QCTYYNDAVDDLLHY. The N-terminal amino acid sequence of the protein showed sequence similarity with the metalloprotease from SM6 and Serratis HR-3.Our results suggested that the factor primarily responsible for proteolytic activity toward casein was a zinc dependent metalloprotease.Based on the high homologous N-terminal amino acid sequence between the protein and metalloprotease, the mature peptide gene of the protease was amplified from total DNA of Serratia nematodiphila by polymerase chain reaction. After cloned into PMD18-T vector, the coding region of mature peptide gene of insecticidal protein has 1398 bp in length and encodes 465 amino acids. Homologous analysis shows there are 98% and 98% homology with those of metalloprotease from SM6 in nucleotides sequence and amino acids sequence, respectively. And the predicted isoelectric point (pI) was 4.57 and the molecular weight was 50642.92 Da that matched well with the observed molecular weight from SDS-PAGE.The optimum pH value for the enzyme was 8.0 and stable in pH 6.0-11.0 for 1 hour. The optimum temperature for the enzyme was about 35℃and stable at temperature below 43℃for 1hour.The Michealis-Menton (Km) was 6.10mg/mL with casein as its substrate.Chelator of divalent cation EDTA,1-10phenanthroline and non-ionic surfactant Tween 20, Tween 40, and Triton X-100 displayed a significant inhibitory effect on the enzyme activity while anionic surfactant SDS did not remarkable inhibit it, After 1 hour in 37℃1mM SDS, the activity of 5. nematodiphila proteases retained 77.6%.Zn²⁺,Cu²⁺can inhibitor the enzyme in different concentration, but Ni²⁺,Mg²⁺,Co²⁺, Ba²⁺,Fe³⁺ can activate the enzyme in the concentration no more than 5 mmol.Mg²⁺,Ca²⁺, Fe²⁺ have no significant effect on the protease activity in variously concentration,but all the ions can inhabit the protease activity in the concentration of 30mmol. All of this indicated that the metal ions very important for preserving structure stabity and protease activity of the protein,so the protein shoud be metalloprotease.