Clone And Prokaryotic Expression Of Stenotrophomonas Maltophilia D2 Alkaline Phosphatase

Objective We obtained a protein-hyperproducing Stenotrophomonas maltophilia strain from the digestive tract of Perinereis aibuhitensis Grube. After the protein purified, we sequenced its amino acid in order to get the coding-sequence by bioinformatics and Molecular Biology technology. We also expressed the coding-sequence in BL-21 to know more about the secretion and expression mechanism of the D2. Methods The purified D2 alkaline phosphatase was digested by enzymes, and then sequenced by ESI-MS/MS. We blasted those six amino acid fragments, and found that those fragments had high identities of the stenotrophomonas maltophilia K279a and R551-3.The phosphatase gene was amplified by the method of PCR with the primer designed according to the K279a and R551-3 who had higher Identities. Then we conjuncted the PCR product to T-vector and sequenced.We also expressed the gene which verification by sequencing in Escherichia coli BL21 (DE3) by yielding hybrid plasmid pET-32a, induced with IPTG, and identify the result by Western Blot. We also used the 48h zymogenic supernatant to detect the alkaline phosphatase activity. Results We got a 1438 bp gene sequence of the stenotrophomonas maltophilia D2, the phosphatase gene contained a single ORF spanning 1233 bp, and its GeneBank number was GQ413951. We translated the ORF into amino acid sequence, and compared it with the stenotrophomonas maltophilia K279a and R551-3alkaline phosphatase. Their identities were 68.5% and 73.6%. We also compared the phosphatase amino acid sequence with the amino acid fragments which sequenced by ESI-MS/MS, the identities were 100%,100%,63.2%,77.8%,88.9% and 42.1%. The ORF encoded a 66KD protein. Western Blot indicated that the recombinant protein is the D2 alkaline protease. We also found the sequence of the first 20 amino acid in the phosphatase amino acid acted as signal peptide, which may play an important role in the proteinl secretion. The 48h zymogenic supernatant had alkaline phosphatase activity, the activities was 18.79. Conclusion we gained the gene of D2 alkaline phosphatase, and successful expressed it in Escherichia coli. The protein had phosphatase activity, it might be a new source of phosphatase of marine microorganisms, and a new expression system.