Characterization of a robust serine protease from Bacillus subtilis K‐1 |
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| Authors: | Satbir Singh Puneet Gupta Bijender K. Bajaj |
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| Affiliation: | School of Biotechnology, University of Jammu, Jammu (J&K), India |
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| Abstract: | Process apt microbial proteases due to their wide range industrial applications have become the focus of intense scientific research during recent years. Considering the hostile process milieu, the proteases intended for application must be robust enough to withstand the extremes of temperature and pH, and presence of organic solvents and other potential enzyme inhibitors. Current study presents the characterization of a robust protease from a previously isolated bacterium Bacillus subtilis K‐1 (BSK‐1). Purification of BSK‐1 protease (5.21‐fold) was achieved to homogeneity by salt (ammonium sulfate) precipitation, and ion‐exchange (diethyl‐aminoethyl‐sephadex) and size exclusion chromatography (Sephadex G‐100). Molecular weight of BSK‐1 protease was determined by SDS‐PAGE analysis (42 kDa). Though the optimum temperature and pH for BSK‐1 protease activity was 50 °C and 10, respectively, but, the protease exhibited remarkable activity and stability over elevated temperatures (60–80 °C) and a broad pH range (pH 7–11). Protease showed resistance towards several organic solvents/other potential enzyme inhibitors. Drastic activity loss in presence of phenylmethylsulfonyl fluoride indicated that the enzyme is a serine protease. Kinetic parameters (Km and Vmax) for BSK‐1 protease were found to be 0.14 mg ml?1 and 1176 mg min?1, respectively. Putative amino acid sequence of BSK‐1 protease (derived from nucleotide sequence of protease gene) suggested that the enzyme belonged to peptidases S8/S53 super family with multidomain of S8. BSK‐1 protease being stable under harsh conditions may serve a model system for understanding the molecular basis of stability, and may help designing novel proteases that are suitable for industrial applications. |
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| Keywords: | Bacillus subtilis characterization chromatography protease purification |
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