Molecular cloning, overexpression and characterization of a new thiol-dependent, alkaline serine protease with destaining function and fibrinolytic potential from Marinobacter aquaeolei MS2-1

Abstract

Abstract Proteolytic marine bacterium designated as strain MS2-1 was isolated from the deep-sea sediment of Bay of Bengal. Strain MS2-1 was taxonomically identified as Marinobacter aquaeolei on the basis of 16S rRNA gene sequence homology analysis. A new alkaline serine protease gene (1,086 bp) was delineated, cloned into pET-28a-(+) vector and overexpressed in Escherichia coli. The enzyme showed 90% amino acid sequence identity towards subtilisin-like protease from a Pseudoalteromonas sp. AS-11. The three-dimensional homology model predicted the active site residues that may be responsible for the proteolytic activity. Molecular weight of the purified protein was calculated as 39 kDa. The enzyme exhibited the stability within a wide range of pH (7.0-12.0) and temperature (40-70°C). Maximum enzyme activity was observed at pH 8.0 and 50°C. The purified enzyme showed stability in the presence of metal ions, solvents, surfactants and detergents. The loss of activity with PMSF and the 3-fold increase of activity with DTT suggested the thiol-dependent nature of this serine protease enzyme. Results reported in this study also suggested that the new alkaline serine protease produced by the strain MS2-1 can be used as an efficient blood stain remover in detergent industries and as a thrombolytic agent in biomedical applications.