The present study was undertaken (a) to formulate an efficient and cost effective medium for the production of proteolytic enzyme(s), and (b) to isolate, purify and characterize new proteolytic enzyme(s) from an indigenous strain of Bacillus subtilis by employing ammonium sulphate fractionation, anion exchange chromatography and cation exchange FPLC.
The work presented here reports the primary structure of a variant of subtilisin from Bacillus subills strain RT-5. Successive fractionations and chromatographies of cell free troth resulted in the isolation of a purified alkaline serine protease subsequently named subtilisin RT-5. Separation of alkaline and neutral proteases on anion exchanger led to the realization that this approach provides an efficient means of separating the two proteases from each other.
The complete primary structure of subtilisin RT-5 has been established by a combination of direct sequence analysis of the intact protein, digestion and peptide analysis and DNA sequence analysis of PCR-produced fragment corresponding to the segment not recovered in the protein digest. The enzyme consists of 275 amino acid residues. It shows 97% homology to mesentericopeptidase with 5 exchanges and 87% homology to subtilisin Novo with 40 amino acid replacements.
Though the physico-chemical properties 01 subtilisin RT-5 are similar to those of subtilisin Novo, its catalytic efficiency has been found to be 40 fold higher than that of subtilisin Novo. The catalytic prowess of subtilisin RT-5 might be due to the amino acid replacements in the region around the active site His64.
The isolated enzyme is closely related to mesentericopeptidase, but it is important to note that this closest homologue is thermolabile suggesting that the replacements of Ala by Ser at positions 85, 89 & 183, and Ser by Ala and Asn at positions 88 & 259 respectively in subtilisin RT-5 might be responsible for its thermostability.
The enzyme shows preference toward hydrophobic residues like valine and phenylalanine at P4 position which might be due to the replacement of Ser130 by Thr130 at the enterence of the P4 binding pocket.