Study Of The Native Promoter Of Alkaline Protease Gene From Bacillus Pumilus

Bacillus pumilus UN-31-C-42 is a strain which produces alkaline protease with dehairing function. The native promoter of alkaline protease gene, Papr, was cloned by TAIL-PCR from B. pumilus UN-31-C-42. Through blast analysis, the sequence of promoter Papr was confirmed and the conserved sequences were deduced. The promoter was 378 bp in length, and a phosphoglycerate mutase gene was located at the upstream region of promoter Papr. Deletion analysis of Papr revealed that the promoter of 160 bp could initiate the transcription of alkaline protease gene.The alkaline protease gene, including promoter, the coding region of prepeptide, propeptide and mature peptide, and terminator, was inserted into pSUGV4, an Escherichia coli-Bacillus subtilis shuttle vector. The recombinant plasmid pSUBpWAp was transformed into E. coli JM109, B. subtilis WB600 and B. pumilus UN-31-C-42, respectively.Expression of alkaline protease gene in pSUBpWAp in E. coli JM109 did not result in detectable extracellular and intracellular protease activity. But it expressed active extracellular protease in B. subtilis WB600 and B. pumilus UN-31-C-42. The enzymatic activity of recombinants in B. subtilis WB600 was 466.5 U/ml. It was twice more than that of plasmid pSUBpAp which was under the control of promoter Bp53. The enzymatic activity of pSUBpWAp in B. pumilus UN-31-C-42 achieved 3960 U/ml, but it was still lower than the host strain.The 16s rDNA was cloned from genome of B. pumilus UN-31-C-42 by PCR. The fragment of 16s rDNA was inserted into vector pMD18-T, constructing recombinant plasmid pMD16s. The fragments of alkaline protease gene and kanamycin-resistant gene were inserted into 16s rDNA in pMD 16s. The recombinant will be integrated in the chromosome of B. pumilus UN-31-C-42 to increase the yield of alkaline protease.