Igentification Of A Pseudomonas Aeruginosa SP. DES-3 Form Marine And The Effect Of Alkaline Protease-Producing By A Gene Of AP02

AlkalineOcean is the largest ecosystem in earth, and the microbial resources in the marine unique environment are different far away from the others natural eco-systems. Besides, the biomass in seabed ooze accounted for more than 10%-30% of the total biomass in earth. Alkaline proteases have been found widely in bacteria, fungi and actinomycetes. And they have also applied in many areas of leather, silk, food, medicine, and biochemistry, especially the application of detergent industry. Screening high-yield alkaline protease strains from marine environments has become a hot spot.First, the study focused on a protease-producing strain DES-3 using a protease activity screening strategy with skimmed-milk from the marine tunnel sludge in Yingkou city of Liaoning Province of China. The DES-3 strain had been identified as the genus of Pseudomonas aeruginosa, and being named a Pseudomonas aeruginosa sp. DES-3 based on the microbial morphological characterization, physiological and biochemical properties,16S rDNA gene identification and phylogenetic tree analysis results. The extracellular protease was partially purified using ammonium sulfate precipitation and ultrafiltration method. The purified protease was found to be stable between 4-55? and displayed a maximum activity at 55?.The enzyme exhibited pH stability in a wide range of 7-9.0 with optimum at 9.0. The enzyme activity was increased marginally in presence of Fe²⁺ and Ca²⁺ and decreased in presence of Zn²⁺, Cu²⁺, Fe3+, Pb²⁺ and Ni²⁺. PMSF completely inhibited the activity, suggesting that it belongs to serine protease.Furthermore, a gene ap02 of producing alkaline protease, which had been identified from an alkaline polluted soil metagenomics library. The total length of the gene was about 1146 base pairs, encodes a polypeptide chain composed of 381 amino acids, and with a molecular weight of about 39.46 kDa, and the isoelectric point is 8.91. At the amino acid level, it shared a similarity of 100% with peptidase from Salinibacillus aidingensis ?GenBank accession number: WP₀44156525.1?. However, the possible peptidase was annotated with microorganism genome sequence, and no further biochemical characterization was performed.The ap02 gene was amplified by PCR, and the product was ligated into the expression vector of pET-32a?+?. After transferring the recombinant plasmid DNA into Escherichia coli BL21 ?DE3?pLysS competent cells. The optimal pH value and optimal temperature for enzyme activity was 9.0 and 65?, respectively. Its activity was stable at pH 7.0-9.0 and stable below 45?. The activity of AP02 protein could be prompted by Ca²⁺ and Mn²⁺ and inhibited by Fe²⁺, Cu²⁺, Ba²⁺ and Zn²⁺. PMSF and EDTA strongly inhibited the activity, which indicated it was dependent in metal ions and belonged to the serine protease family.Gene ap02 had ligated with a widely host expression vector of pMP2444 and pBBR1MCS-5. They were expressed in the wild strain Pseudomonas aeruginosa sp. DES-3 that was mediated with 0.3 M sucrose solution. The study suggestions that the enzyme activity of two recombinant strains were slightly lower than that of the wild strain at the same temperature. But the optimum temperature of recombinant strains DES-3/pBBRlMCS-5-ap02 was 60?. The final optimal temperature had increased 5? than that of wild type strain of Pseudomonas aeruginosa sp. DES-3.This study breaks the general methods of strain transformation, attempting to express the protein gene ap02 in the wild strain DES-3. As a result, the optimum temperature has been changed, which will show a technical reference for the genetic modification of the wild type strain and the potential industrial application.