Cloning, expression, purification and characterisation of serine alkaline protease from Bacillus subtilis RD7

Proteases (E.C 3.4) are enzymes that break peptide bonds between amino acid groups of proteins. Bacillus subtilis RD7, an extracellular protease producer was genetically characterised, B. subtilis genomic DNA was isolated, oligonucleotide primers specific to serine alkaline protease gene of B. subtilis were designed and its PCR amplification was done. The purified PCR product and pET15b vector were subjected to restriction digestion with Nde1 and BamH1 and transformed into Escherichia toll DH5 alpha competent cells. The recombinant expression of serine alkaline protease gene studied by inducible expression and analysis by SDS-PAGE, established that the serine alkaline protease protein had an estimated molecular size of 43 kDa. Gene sequencing of the insert from selected recombinant clone showed it to be a 1203bp gene encoding a protein of 400 amino acids. The sequence was blasted and aligned with known serine alkaline protease genes for comparison with their nucleotide and amino acid sequences. This identified major matches with closely related subsp. of B. subtilis. The insert also showed many substitutions (mutations with other sp. of Bacillus which established that serine alkaline protease of B. subtilis RD7 is a novel gene. The phylogenetic analysis of serine alkaline protease gene and its predicted amino acid sequences also validated that serine alkaline protease gene is a novel gene and has been accessioned in GenBank with accession number (MN097797). When expressed in E. coli, the recombinant enzyme was over expressed in the cytoplasm as soluble and active form. The purified enzyme was completely inhibited by PMSF. The enzyme showed maximum activity at pH 10 and 40 'C. It was stable at pH from 6 to 11 and below 70 'C. The aim of this study is to clone serine alkaline protease gene from B. subtilis RD7 (MG255317), its expression in mesophilic E. coli. strain BL21, Purification and characterisation of the expressed protein.