Salt-enhanced processing, proteolytic activity and stability of halophilic thermolysin-like proteinase, salilysin, isolated from a moderate halophile, Chromohalobacter salexigens DSM3043
Ryoichi Tanaka, Shunsuke Yamasaki, Matsujiro Ishibashi, Hiroko Tokunaga, Tsutomu Arakawa, Masao Tokunaga
International Journal of Biological Macromolecules
Moderately halophilic bacterium, Chromohalobacter salexigens DSM3043, has a gene Csal_2537 encoding thermolysin-like M4 proteinase. This gene was cloned to pET expression vectors, resulting in high expression of recombinant proteinase, named as salilysin (salinity-dependent thermolysin-like proteinase), in Escherichia coli cytoplasm. This gene encodes precursor form of salilysin containing 348 amino acid residues (Pro-salilysin) consisting of 55 amino acids pro-sequence and following mature proteinase. Pro-sequence was cleaved three times to form intermediate 1, intermediate 2 and final mature salilysin. The processing rate was greatly accelerated in a salt concentration-dependent manner. Purified inactive mutant Pro-E167A-salilysin was correctly processed by purified mature salilysin, indicating that autolysis and inter-molecular processing occurred in its maturation processes. Proteolytic activity of mature salilysin against both peptide and protein substrates was also enhanced along with the addition of higher concentration of salt, 0–3.2 M NaCl, consistent with its halophilic origin. Mature salilysin was stabilized by ~8 °C in the presence of 1 M NaCl by thermal scanning using circular dichroism. One of the precursor form, intermediate 1, showed ~20 °C higher denaturation temperature than mature form, suggesting rigid and stable structure of this precursor form.
Circular dichroism, Secondary structure, Thermal stability, Chemical stability, Protein folding, Biochemistry