Cloning, Sequencing and Expression of an Organic Solvent Tolerant Protease from Bacillus Pumilus 115B

Five out of the nine isolated bacteria species screened earlier, demonstrated high protease production on Skim Milk Agar and were also to Benzene Toulene Xylene Ethylbenzene (BTEX). Among these, isolate 115b was found to produce the highest protease production. Besides being stable in 25% (v/v) benzene and toluene, protease from isolate 115b was found to be activated by n-dodecane and n-tetradecane by 1.7 and 2.5 folds respectively. Isolate 115b was identified as Bacillus pumilus 115b via biochemical tests and 16S rDNA sequencing analysis. The gene encoding protease of Bacillus pumilus 115b was amplified via polymerase chain reaction (PCR) using consensus primers based on the sequences of alkaline serine protease genes from related species. The complete nucleotide sequence of the protease from Bacillus pumilus 115b was determined. Sequence analysis showed an open reading frame (ORF) of 1149 that encoded a polypeptide of 383 amino acid residues and the calculated protein molecular mass of 39,448 Da. The ORF also encoded a single peptide consisting of 29 residues and a propeptide of 79 residues. The mature protein comprised 275 amino acids with a calculated molecular mass of 27,846 Da. Homology searches revealed that the amino acid residues from B. pumilus 115b protease shared a high homology (90%) with the alkaline serine protease from B. pumilus TYO-67 and B. pumilus UN-31-C-42. The gene coding for an organic solvent tolerant 115b protease gene was cloned into pQE-30 UA expression vector. The recombinant plasmid was then transformed into E. coli M15[pREP4]. The organic solvent tolerant 115b protease gene was successfully expressed by induction with IPTG and was detected by SDS-PAGE analysis with a molecular weight of around 35 kDa. The expression of recombinant E. coli M15[pREP4] was optimized by inducing it with 1.0 mM of IPTG at 4 hour of induction time.

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