Salt-enhanced processing, proteolytic activity and stability of halophilic thermolysin-like proteinase, salilysin, isolated from a moderate halophile, Chromohalobacter salexigens DSM3043.

International Journal of Biological Macromolecules
Ryoichi TanakaMasao Tokunaga

Abstract

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, ...Continue Reading

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