Purification and stability characteristics of an alkaline serine protease from a newly isolated Haloalkaliphilic bacterium sp. AH-6

  • M. S. Dodia
  • C. M. Rawal
  • H. G. Bhimani
  • R. H. Joshi
  • S. K. Khare
  • S. P. Singh
Original Paper


An alkaline protease secreting Haloalkaliphilic bacterium (Gene bank accession number EU118361) was isolated from the Saurashtra Coast in Western India. The alkaline protease was purified by a single step chromatography on phenyl sepharose 6 FF with 28% yield. The molecular mass was 40 kDa as judged by SDS-PAGE. The enzyme displayed catalysis and stability over pH 8–13, optimally at 9–11. It was stable with 0–4 M NaCl and required 150 mM NaCl for optimum catalysis at 37 °C; however, the salt requirement for optimal catalysis increased with temperature. While crude enzyme was active at 25–80 °C (optimum at 50 °C), the purified enzyme had temperature optimum at 37 °C, which shifted to 80 °C in the presence of 2 M NaCl. The NaCl not only shifted the temperature profile but also enhanced the substrate affinity of the enzyme as reflected by the increase in the catalytic constant (K cat). The enzyme was also calcium dependent and with 2 mM Ca+2, the activity reached to maximum at 50 °C. The crude enzyme was highly thermostable (37–90 °C); however, the purified enzyme lost its stability above 50 °C and its half life was enhanced by 30 and sevenfold at 60 °C with 1 M NaCl and 50 mM Ca+2, respectively. The activity of the enzyme was inhibited by PMSF, indicating its serine type. While the activity was slightly enhanced by Tween-80 (0.2%) and Triton X-100 (0.05%), it marginally decreased with SDS. In addition, the enzyme was highly stable with oxidizing-reducing agents and commercial detergents and was affected by metal ions to varying extent. The study assumes significance due to the enzyme stability under the dual extremities of pH and salt coupled with moderate thermal tolerance. Besides, the facts emerged on the enzyme stability would add to the limited information on this enzyme from Haloalkaliphilic bacteria.


Haloalkaliphiles Alkaline protease Purification Salt-dependent thermostability 


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Copyright information

© Society for Industrial Microbiology 2007

Authors and Affiliations

  • M. S. Dodia
    • 1
  • C. M. Rawal
    • 1
  • H. G. Bhimani
    • 1
  • R. H. Joshi
    • 1
  • S. K. Khare
    • 2
  • S. P. Singh
    • 1
  1. 1.Department of BiosciencesSaurashtra UniversityRajkotIndia
  2. 2.Department of ChemistryIndian Institute of TechnologyNew DelhiIndia

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